Methods, compositions, and devices for supplying dietary fatty acid needs

ABSTRACT

Nutritional formulas comprising long-chain polyunsaturated fatty acids (LC-PUFAs) are provided, along with methods and devices for preparing and/or administering nutritional formulas. In some embodiments, a percentage of the LC-PUFAs in the nutritional formula are in the form of monoglycerides and/or free fatty acids. In some embodiments, the nutritional formulas do not comprise added lipase. Also provided are methods for providing nutrition to a subject, methods for improving fat absorption, methods for improving cognitive ability, methods for preventing chronic lung disease, and methods for reducing the length of time a patient requires total parenteral nutrition.

This application is a continuation of U.S. application Ser. No.15/240,596, filed Aug. 18, 2016, which is a continuation of U.S.application Ser. No. 14/378,856, filed Aug. 14, 2014, which is aNational Stage Application of PCT/US2013/026063, filed Feb. 14, 2013,which claims priority under 35 U.S.C. §119 to U.S. ProvisionalApplication No. 61/600,207, which was filed on Feb. 17, 2012, and toU.S. Provisional Application No. 61/719,173, which was filed on Oct. 26,2012, each of which is incorporated by reference herein in its entirety.

Long-chain fatty acids are critical to human health and development.Long-chain fatty acids that are consumed in the diet are primarily inthe form of triglycerides (TGs), in which three long-chain fatty acidsare bound to a glycerol molecule via ester linkages. Absorption oflong-chain triglycerides first requires the enzymatic action of lipases,(e.g. pancreatic lipase), which digest triglycerides through hydrolysis,breaking them down into monoglycerides and further into free fattyacids. Once available, these monoglycerides and free fatty acids areabsorbed by endothelial cells in the small intestine, where they undergoreesterification, followed by transport to the liver and ultimately totissues in the body for various physiological purposes. D. Kasper etal., Harrison's Principles of Internal Medicine 16^(th) Ed. (2004).While medium chain triglycerides can be absorbed across the intestinallumen, long-chain triglycerides cannot, therefore, pancreatic lipase isessential for proper long-chain fatty acid hydrolysis and absorption. C.Jensen et al., Am. J. Clin. Nutr. 43:745-751 (1986). However, somepeople are unable to adequately breakdown long-chain triglycerides,e.g., patients suffering from compromised pancreatic output,malabsorption or pancreatic insufficiency, and as a result, may sufferfrom absorption of fatty acids that is inadequate to maintain health.

Commercially available lipase supplements may be added to the diet toimprove hydrolysis of long-chain triglycerides. However, for a number ofreasons, lipase supplements will not necessarily solve the problem ofpoor fatty acid absorption in all people suffering from reduced abilityto break down long chain triglycerides or otherwise in need of receivingelemental fatty acids. For example, most commercial lipase supplementsare made from animal pancreatic lipase, which is known to havesignificantly reduced stability below pH 7. See, e.g., US2010/0239559,D. Kasper et al., Harrison's Principles of Internal Medicine 16^(th) Ed.(2004). By the time such lipases pass through the stomach, significantamounts are likely to have been inactivated. Further, not all lipaseswork to the same degree for hydrolysis of a given long-chain fatty acid,indicating lipase specificity is an important consideration. R. Jensenet al., Lipids 18(3):239-252 (1983). And in some populations withpancreatic insufficiency, nutritional formulas are tightly regulated,such as in pre-term infants or in patients in intensive care units. Forthese controlled populations, it may not be desirable or feasible tosupplement already-approved formulas with additional ingredients.Moreover, although many fatty acid supplemented formulas may containmedium-chain triglycerides, there is a distinct medical benefit todietary intake of long-chain fatty acids. Thus, there is a need forimproved methods of enhancing hydrolysis of long-chain triglycerides.

Proper hydrolysis of long-chain polyunsaturated triglycerides(TG-LCPUFA) is particularly important for several reasons. Long-chainpolyunsaturated fatty acids (LC-PUFAs) are critical for neural andretinal development. Moreover, some are considered “essential fattyacids,” meaning that humans cannot synthesize them and must obtain themfrom dietary sources. The principal dietary source for n-3 LC-PUFAsdocosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is theirprecursor, alpha-linolenic acid (ALA), which is an essential fatty acid.Endogenous enzymes, however, are highly inefficient at converting ALA toDHA and EPA. According to an official statement by the InternationalSociety for the Study of Fatty Acids and Lipids (ISSFAL), the conversionof ALA to DHA is about 1% in infants and considerably lower in adults.Brenna et al., Prostaglandins Leukot Essent Fatty Acids, 80(2-3):85-91(2009). Thus, although DHA and EPA are not essential fatty acids per se,dietary sources of DHA and EPA are important. The principal dietarysource for the n-6 LC-PUFA arachidonic acid (ARA or AA) is linoleic acid(LA), which is an essential fatty acid.

Embodiments of the invention solve these various problems by (i)providing lipases that are surprisingly more efficient than others athydrolyzing certain long-chain triglycerides and esters, such as, e.g.,long-chain polyunsaturated triglycerides and esters (ii) providing anutritional formula, such as, e.g., a medical nutritional formula or aninfant formula, comprising pre-hydrolyzed components (i.e.,monoglycerides and/or free fatty acids) of LC-PUFA triglycerides,LC-PUFA fatty acid esters, and/or other long-chain triglycerides orlong-chain fatty acid esters, (iii) providing methods of producing suchnutritional formula, including methods in which a formula containingLC-PUFA triglycerides, LC-PUFA fatty acid esters, and/or otherlong-chain triglycerides or long-chain fatty acid esters is temporarilyexposed to lipase and (iv) providing devices designed to providenutritional formulas comprising monoglycerides and/or free fatty acids,e.g., LC-PUFA triglycerides and/or LC-PUFA fatty acid esters. Inembodiments in which the formula is temporarily exposed to the lipaseand the lipase is removed or separated from the formula prior toingestion, the invention provides the advantage of ensuring breakdown ofLC-PUFA triglycerides, LC-PUFA fatty acid esters, and/or otherlong-chain triglycerides or long-chain fatty acid esters withoutrequiring ingestion of exogenous lipase.

Accordingly, some embodiments of the invention provide a nutritionalformula. In some embodiments, the nutritional formula comprisesLC-PUFAs. In some embodiments, more than 2% of the total LC-PUFAs are inthe form of monoglycerides and free fatty acids, i.e. less than 98% ofthe total LC-PUFAs are in triglyceride or ester form. In someembodiments, the LC-PUFA monoglycerides and free fatty acids comprisemore than 2.5%, more than 3%, more than 4%, more than 5%, more than 6%,more than 7%, more than 8%, more than 10%, more than 12%, more than 15%,more than 20%, more than 25%, more than 30%, more than 40%, more than50%, or more than 75% of the total LC-PUFAs in a nutritional formula. Incertain embodiments, the ratio of LC-PUFA monoglycerides and free fattyacids to triglycerides and esters is at least 0.08:1, at least 0.09:1,at least 0.1:1, at least 0.25:1, at least 0.5:1, at least 1:1, at least2:1, at least 3:1, at least 4:1, at least 5:1, at least 8:1, at least10:1, or at least 20:1.

In certain embodiments, the nutritional formula is formulated foradministration to premature infants. Other nutritional formulasencompassed by the invention are formulated for infants, toddlers,children, or adults who have a reduced ability to hydrolyze LC-PUFAtriglycerides, LC-PUFA fatty acid esters, and/or other long-chaintriglycerides or long-chain fatty acid esters, or who simply needadditional elemental dietary LC-PUFAs and/or other long-chain fattyacids. In some embodiments, a nutritional formula of the invention isfor a subject who is less than 1 year old. In some embodiments, thesubject is between 1 and 4 years old. In some embodiments, the subjectis between 1 and 6 years old.

In certain embodiments, the nutritional formula of the invention is amedical nutritional formula, i.e., a formula that is formulated to beconsumed or administered orally or enterally under medical supervision,such as those distributed through hospitals or pharmacies under aprescription. Typically, a medical nutritional formula is formulated fordietary management of a specific medical disorder, disease, or abnormalcondition, for which there are distinctive nutritional requirements. Amedical nutritional formula must have “Generally Recognized As Safe”status and comply with FDA regulations that pertain to labeling, productclaims, and manufacturing.

In some embodiments, the nutritional formula does not contain addedlipase. In other embodiments, the nutritional formula contains a lipase.In some embodiments, the lipase is selected from Chromobacteriumviscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopusoryzae lipases.

In some embodiments, the nutritional formula comprises EPA, DHA, ARA,LA, and/or ALA.

Because free polyunsaturated fatty acids are unstable and rapidlydegrade, the invention also provides convenient and effective methods ofpreparing the nutritional formulas of the invention shortly beforeingestion by a subject. In certain embodiments, the method comprisesexposing a liquid nutritional composition comprising LC-PUFAtriglycerides, LC-PUFA fatty acid esters, and/or other long-chaintriglycerides and/or esters of long-chain fatty acids to a lipase priorto ingestion by a person in need of additional dietary LC-PUFAs and/orother long-chain fatty acids. In some embodiments, the liquidnutritional composition is exposed to lipase for at least one minute, atleast 2 minutes, at least 3 minutes, at least 5 minutes, at least 8minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes,at least 45 minutes, or at least 60 minutes prior to ingestion. In someembodiments, the liquid nutritional composition is exposed to lipase forno more than one minute, no more than 2 minutes, no more than 3 minutes,no more than 5 minutes, no more than 8 minutes, no more than 10 minutes,no more than 15 minutes, no more than 30 minutes, no more than 45minutes, or no more than 60 minutes prior to ingestion. In someembodiments, the liquid nutritional composition is exposed to lipase forno more than 24 hours. In certain embodiments, the lipase is selectedfrom Chromobacterium viscosum, Pseudomonas fluorescens, Burcholderiacepacia, and Rhizopus oryzae lipases. In certain embodiments, the lipasemay be removed from the nutritional formula prior to ingestion. In otherembodiments, the liquid nutritional composition comprising LC-PUFAtriglycerides, LC-PUFA fatty acid esters, and/or other long-chaintriglycerides and/or esters is exposed to lipase immobilized to a solidsupport prior to ingestion. In some embodiments, the lipase isimmobilized to the solid support by covalent binding, ionic binding, orcrosslinking. In certain embodiments, the immobilized lipase isencapsulated within or attached to a permeable membrane.

Another aspect of the invention is a method of providing nutrition to asubject in need of dietary LC-PUFAs and/or other long-chain fatty acids,such as people suffering from a reduced ability to break down long-chaintriglycerides or long-chain fatty acid esters in the gut, peoplesuffering from pancreatic insufficiency, people suffering frommalnutrition, and people who have been receiving total parenteralnutrition, by administering a formula of the invention. In someembodiments, the subject is a premature infant. In other embodiments,the subject is a term infant or toddler. In certain embodiments, thesubject is over the age of 50, over the age of 60, or over the age of70. In some embodiments, the subject is suffering from pancreaticinsufficiency. In other embodiments, the formula is administered througha feeding tube. In some embodiments, the nutritional formula of theinvention are administered to improve cognitive ability in a person ofany age, to prevent chronic lung disease in a pre-term infant, toenhance the neurological development of a pre-term infant, or to treator prevent a number of other conditions associated with improvement fromincreased intake of long-chain fatty acids, such as, e.g., EPA, DHA,ARA, LA, and ALA. Such conditions include but are not limited toAlzheimer's disease, bipolar disorder, depression, sepsis, acuterespiratory stress, wound healing, cancer, cardiovascular disease,stroke, Parkinson's disease, schizophrenia, diabetes, multiplesclerosis, malnutrition, impaired GI function, and chronic inflammatorydiseases such as rheumatoid arthritis, systemic lupus erythematosus, andinflammatory bowel disease.

Another embodiment of the invention provides a method for reducing thetime a patient needs total parenteral nutrition by administering anutritional formula of the invention. As a result, such patients areexposed to a reduced risk of gut atrophy and other complicationsassociated with extended (more than 24 hours) total parenteralnutrition. Such methods may be used to shorten the recovery time ofpatients suffering from impaired GI function, such as, e.g.,malabsorption, short bowel syndrome, IBD, pancreatic insufficiency,malnutrition before or after surgery, chemo- or radiotherapy, or othercauses of malnutrition, cancer, wouns, and pressure ulcers. Suchpatients may receive the nutritional formula of the invention vianasogastric tube. This feeding method may be advantageous in situationswhere the patient suffers from altered gut motility, impaired pancreaticenzyme secretion due to Systemic Inflammatory Response Syndrome, orother conditions that result in impaired cleavage and absorption ofLC-PUFA triglycerides, LC-PUFA fatty acid esters, and/or otherlong-chain triglycerides or esters of long-chain fatty acids. In analternate embodiment, where it is advantageous to bypass the stomach,the nutritional formula of the invention may be administered bynasojejunal tube. Other types of feeding apparatus may also be used todeliver the formulas of the invention.

Since healthy subjects may also benefit from increased absorption ofLC-PUFAs, e.g., by reducing the risk of cardiovascular disease.Accordingly, in some embodiments, the invention provides methods ofimproving fat absorption in a healthy subject, comprising feeding to thesubject a nutritional formula of the invention.

The invention further provides devices for preparing the nutritionalformulas of the invention. In some embodiments, the device comprises achamber containing at least one lipase, wherein the chamber is capableof holding a liquid nutritional composition so that it is exposed to thelipase. In some embodiments, the lipase in the container is immobilizedto the inner surface of the container. In other embodiments, the lipaseis immobilized to a support within the chamber. In some embodiments, thedevice comprises a chamber consisting of a permeable membrane andcomprising immobilized lipase within the chamber, such that the liquidnutritional composition may flow through the permeable membrane and comein contact with the lipase, but the lipase cannot pass through thepermeable membrane. In some embodiments, the lipase contained within thechamber of a device of the invention is a microbial lipase. In someembodiments, the lipase is selected from bacterial lipases. In someembodiments, the lipase is selected from Chromobacterium viscosumlipase, Pseudomonas fluorescens lipase, Burcholderia cepacia lipase, andRhizopus oryzae lipase. In some embodiments, the lipase is selected fromChromobacterium viscosum lipase, Pseudomonas fluorescens lipase, andRhizopus oryzae lipase. In some embodiments, the lipase is Rhizopusoryzae lipase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a device and method for providing nutrition to aninfant, according to certain embodiments.

FIG. 2A illustrates a device for treating formula, according to certainembodiments.

FIG. 2B illustrates a device for treating formula, according to certainembodiments.

FIG. 2C illustrates a device for treating formula, according to certainembodiments.

FIG. 3A illustrates a device for treating formula, according to certainembodiments.

FIG. 3B illustrates a device for treating formula, according to certainembodiments.

FIG. 3C illustrates a device for treating formula, according to certainembodiments.

FIG. 4A illustrates a device for treating formula, according to certainembodiments.

FIG. 4B illustrates a device for treating formula, according to certainembodiments.

FIG. 5A illustrates a device for treating formula, according to certainembodiments.

FIG. 5B illustrates a device for treating formula, according to certainembodiments.

FIG. 6A is a photograph of a vial of Rhizopus oryzae lipase immobilizedon polymer beads.

FIG. 6B illustrates a device for treating and administering nutritionalformula according to certain embodiments.

FIG. 6C illustrates a device for treating and administering nutritionalformula according to certain embodiments.

FIG. 6D illustrates a close up of the device depicted in FIG. 6C.

FIG. 7 depicts the hydrolysis of DHA triglyceride by Rhizopus oryzae(RO) lipase.

FIG. 8 depicts the hydrolysis of ARA triglyceride by Rhizopus oryzaelipase.

FIG. 9A illustrates a device for treating formula, according to certainembodiments.

FIG. 9B illustrates a device for treating formula, according to certainembodiments.

FIG. 9C illustrates a device for treating formula, according to certainembodiments.

FIG. 10A illustrates a device for treating formula, according to certainembodiments.

FIG. 10B illustrates a device for treating formula, according to certainembodiments.

FIG. 10C illustrates a device for treating formula, according to certainembodiments.

FIG. 11A illustrates a device for treating formula, according to certainembodiments.

FIG. 11B illustrates a device for treating formula, according to certainembodiments.

FIG. 11C illustrates a device for treating formula, according to certainembodiments.

FIG. 12 illustrates a device for treating formula, according to certainembodiments.

FIG. 13A illustrates a device for treating formula, according to certainembodiments.

FIG. 13B illustrates a device for treating formula, according to certainembodiments.

FIG. 14 illustrates a device for treating formula, according to certainembodiments.

FIG. 15 illustrates a device for treating formula, according to certainembodiments.

FIG. 16 illustrates a device for treating formula, according to certainembodiments.

FIG. 17A illustrates a device for treating formula, according to certainembodiments.

FIG. 17B illustrates a partial cut-away view of a device for treatingformula, according to certain embodiments.

FIG. 18 illustrates a device for treating formula, according to certainembodiments.

FIG. 19 illustrates a partial cut-away view of a device for treatingformula, according to certain embodiments.

FIG. 20 illustrates a partial cut-away view of a device for treatingformula, according to certain embodiments.

FIG. 21 illustrates a partial cut-away view of a device for treatingformula, according to certain embodiments.

FIG. 22A shows stool weight in EPI pigs during the control week (“Contweek,” during which pigs were fed TG-LCPUFA fortified infant formula)vs. the treatment week (“Treat week,” during which pigs were fed thesame fortified formula either non-hydrolyzed (“CONT”), pre-hydrolyzedwith Chromobacterium viscosum lipase (“CV”), or pre-hydrolyzed withRhizopus oryzae lipase (“RO”)). FIG. 22B shows total fecal fat contentover the last 3 days of the treatment week in the same 3 groups of pigs,and FIG. 22C shows the coefficient of fat absorption (% CFA).

FIGS. 23A-23C show the levels of ARA (FIG. 23A), EPA (FIG. 23B), and DHA(FIG. 23C) in the faeces of EPI pigs fed non-hydrolyzed formula(“CONT”), formula pre-hydrolyzed with CV lipase (“CV”), or formulapre-hydrolyzed with RO lipase (“RO”). Asterisks indicate p<0.001.

FIGS. 24A and 24B show levels of ARA (FIG. 24A) and DHA (FIG. 24B) inthe plasma of EPI pigs following 7 days of feeding with non-hydrolyzedformula (“CONT”), formula pre-hydrolyzed with CV lipase (“CV”), orformula pre-hydrolyzed with RO lipase (“RO”). Asterisks indicate p<0.05.

FIGS. 25A and 25B show levels of ARA and DHA in the retina (FIG. 25A)and adipose tissue (FIG. 25B) of EPI pigs following 7 days of feedingwith non-hydrolyzed formula (“CONT”), formula pre-hydrolyzed with CVlipase (“CV”), or formula pre-hydrolyzed with RO lipase (“RO”).Asterisks indicate p<0.05.

FIGS. 26A and 26B show levels of ARA and DHA in the heart (FIG. 26A) andkidney tissue (FIG. 26B) of EPI pigs following 7 days of feeding withnon-hydrolyzed formula (“CONT”), formula pre-hydrolyzed with CV lipase(“CV”), or formula pre-hydrolyzed with RO lipase (“RO”). Asterisksindicate p<0.05.

FIGS. 27A-27D show percent hydrolysis of DHA and ARA in Enfalac formulawith 100 mg (FIG. 27A), 500 mg (FIG. 27B), 1000 mg (FIG. 27C), or 2000mg (FIG. 27D) of immobilized RO lipase.

FIGS. 28A and 28B show percent hydrolysis of DHA (FIG. 28A) and ARA(FIG. 28B) with RO lipase or pancreatin.

FIG. 29 depicts the study design for the 6-week pig study described inExample 10.

FIGS. 30A and 30B show levels of ARA (FIG. 30A) and DHA (FIG. 30B) inerythrocytes collected from healthy pigs fed TG-LCPUFA-fortified infantformula (“Healthy”), pigs with surgically-induced exocrine pancreaticinsufficiency fed TG-LCPUFA-fortified infant formula (“EPI”), and pigswith surgically-induced exocrine pancreatic insufficiency fedTG-LCPUFA-fortified infant formula that had been pre-hydrolyzed byimmobilized RO lipase (“EPI+iRO”).

LONG-CHAIN POLYUNSATURATED FATTY ACIDS

Long-chain polyunsaturated fatty acids (LC-PUFAs) are hydrocarbon chainscontaining two or more double bonds. Depending on the position of thefirst double bond relative to the methyl terminus, an LC-PUFA can beclassified as an omega-3 (n-3) or omega-6 (n-6) fatty acid. ALA and LAare parent fatty acids of the n-3 and n-6 PUFA families, respectively.They are considered “essential fatty acids,” meaning that humans cannotsynthesize them, but rather, must obtain them through diet. This isbecause mammals lack the ability to introduce double bonds in fattyacids beyond carbon 9 and 10. Blosover et al. Cell Biology: A ShortCourse, John Wiley & Sons, Inc. at 39 (2011). However, humans can makeadditional long-chain PUFAs starting with ALA and LA.

Both ALA and LA are metabolized to generate other long-chain PUFAsthrough a series of desaturation and elongation steps. For example, ALAis metabolized to EPA and ultimately DHA. LA is metabolized to ARA, ann-6 fatty acid. Conversion of ALA to DHA and EPA and LA to ARA however,is relatively inefficient. L. Arterburn et al., Am. J. Clin. Nutr.83(suppl):1467S-1476S (2006). Studies have estimated the conversion ofALA to DHA in humans is less than 5%. B. Anderson and D. Ma, LipidsHealth Dis. 8:33 (2009). The liver contains the most active tissue forconverting ALA to DHA and LA to ARA, and therefore plays a key role inproviding DHA and ARA to less active tissues or organs, such as thebrain. M. Martinez et al., J. Pediatr. 120:S129-S138 (1992).Alternatively, these LC-PUFAs can be consumed directly from the diet.DHA and EPA are found in fish, walnuts, and flaxseed oil, whereas ARA isavailable from animal fat sources, corn oil, soybean oil and sunflowerseed oil.

n-3 Fatty Acids

The n-3 fatty acid DHA is critical to neural and retinal development andfunction. It is the main long-chain PUFA in the neural membrane and isessential for brain function, building of brain circuits, and nerveimpulse transmission. As an integral membrane component, DHA contributesto membrane fluidity which is important for maintaining synapticstructures, neurotransmission, and synaptic plasticity. G. Jicha et al.,Clin. Interv. Aging 5:45-61 (2010). DHA also influences signaling eventsessential to neuron differentiation and survival, and has effects on thelevels and metabolism of neurotransmitters and eicosanoids. The majorityof DHA accumulation in the brain occurs at the beginning of the 3^(rd)trimester throughout the second year of life. It has been demonstratedthat in rodents and primates, an inadequate supply of n-3 PUFA duringthis period results in impaired learning capacity and neurotransmission.M. Martinez et al., J. Pediatr. 120:S129-S138 (1992). Supplementation ofDHA in rats that were previously restricted to a DHA-deficient dietrescues performance in memory and learning tasks. W. Chung et al., J.Nutr. 138(6):1165-1171 (2008). And in a study of healthy adolescentboys, 8 weeks of DHA supplementation significantly increased functionalactivation in the dorsolateral prefrontal cortex during performance ofan activation task compared to placebo. R. McNamara et al., Am. J. Nutr.91:1060-7 (2010). Thus, DHA is considered important not only indevelopment, but in the maintenance of neuronal function.

DHA is also highly concentrated in the retina and has important effectson photoreceptor differentiation and activation of the visual pigmentrhodopsin. H. Lauritzen et al., Prog. Lipid Res. 40:1-94 (2001); M.Clandinin et al., J. Pediatr. 125:S25-32 (1994). An inadequate supply ofDHA early in the development of primates and rodents results in abnormalretinal physiology and reduced visual acuity. M. Reisbick et al., Dev.Psychol. 33:387-395 (1997); J. McCann et al., Am. J. Clin. Nutr.82:281-295 (2005). Similarly, in humans, infants fed formula without DHAfor the first twelve months of life have been shown to have lower visualacuity than infants fed DHA-supplemented formula. E. Birch et al., Am.J. Clin. Nutr. 91(4):848-859 (2010). DHA deficiencies have also beenassociated with fetal alcohol syndrome, attention deficit hyperactivitydisorder, cystic fibrosis, phenylketonuria, unipolar depression,aggressive hostility, and adrenoleukodystrophy. A. Horrocks et al.,Pharmacological Res. 40(3):211-225 (1999).

The benefits of increased intake of DHA and other n-3 fatty acids havebeen described for various diseases, including for example, Alzheimer'sdisease (AD), bipolar disorder (BP), depression, including majordepressive disorder (MDD) and post-partem depression, sepsis, acuterespiratory stress, wound healing, cancer, cardiovascular disease,stroke, Parkinson's disease, schizophrenia, diabetes, multiplesclerosis, and chronic inflammatory diseases such as rheumatoidarthritis, systemic lupus erythematosus, and inflammatory bowel disease.

For example, clinical trials with AD patients have demonstrated that DHAprovides a therapeutic benefit. For a review of studies evaluating theeffect of DHA in AD, see G. Jicha and W. Markesbery, Clin. Interv. Aging5:45-61 (2010). Data from in vitro assays, cell culture systems, andmurine models of AD support a direct role for n-3 PUFAs in amyloidprocessing in the brain. And in amyloid-producing transgenic models ofAD, supplementation with DHA results in lower levels of αδ. M. Oksman etal., Neurobiol. Dis. 23(3):563-572 (2006). In addition to positiveclinical trial data in patients with AD, a large study in healthyelderly people with mild memory complaints showed that subjectsadministered DHA performed better on learning and memory tests after sixmonths compared to those receiving placebo. (Martek Press Release, May4, 2010). Thus, DHA may also play a beneficial role in preventing AD.

Therapeutic use of DHA has also been investigated in patients with BPand MDD. For a review on DHA in BP, see V. Balencia-Martinez et al.,Expert. Rev. Neurother. 11(7):1029-1047 (2011). Due to the difficultiesin assessing DHA levels in brain tissue from human patients, the fattyacid composition in erythrocyte membranes from blood samples has beenevaluated and was found to contain significantly less DHA in patientswith BP and MDD than in healthy controls. R. McNamara et al., J. Affect.Disord. 126(1-2):303-311 (2010). In a post-mortem study, the fatty acidcomposition of the orbitofrontal cortex had significantly lower levelsof DHA in BP patients compared to normal controls. R. McNamara et al.,Psychiatry Res. 160(3):285-299 (2008). And in a 4-month, double-blind,placebo-controlled study, BP patients receiving n-3 fatty acids had asignificantly longer period of remission than the placebo group. A.Stoll et al., Arch. Gen. Psychiatry 56(5):407-412 (1999). These studiesimplicate DHA as therapeutically beneficial in BD and MDD, particularlydue to its mood-stabilizing effects.

DHA has also been shown to benefit patients suffering from other formsof depression. For a review, see A. Logan et al., Lipids Health Dis.3:25-32 (2004). A number of studies have found decreased n-3 levels inthe blood of patients with depression. Similarly, an increase in plasmaDHA was associated with a reduction in women reporting symptoms ofpost-partem depression. Some placebo-controlled studies have found n-3treatment improves depressive systems. For a review on the relationshipbetween n-3 levels and depression, see A. Logan et al., Lipids HealthDis. 3:25-32 (2004).

In sepsis, an enteral diet enriched with EPA, γ-linolenic acid, andantioxidants improved hospital outcomes and reduced mortality inpatients with severe sepsis or septic shock requiring mechanicalventilation. A. Pontes-Arruda et al., Crit. Care Med. 34(9):2325-2333(2006). Similar benefits in ventilator-free days, ICU-free days, reducednew organ dysfunctions, and a decreased mortality rate have beenreported in patients with acute respiratory stress fed a diet enrichedlong-chain PUFAs and antioxidants. J. Gadek et al. Crit. Care Med.27(8):1409-1420 (1999).

N-3 fatty acids are also reported to have beneficial effects in woundhealing. Through altering the lipid microenvironment, n-3 fatty acidsenhance the reconstitution of epithelial cells and may also help toreduce inflammation. D. Ruthig and K. Meckling-Gill, J. Nutr.129:1791-1798 (1999); J. McDaniel et al. Wound Repair Regen.19(2):189-200 (2011).

EPA and DHA have shown protective effects in cancers, such as prostateand breast cancer. The beneficial effects may be due toanti-inflammatory properties, as well as mechanisms that decreaseproliferation and promote apoptosis, such as through downregulation ofNF-κB. For a discussion on n-3 fatty acids in cancer, see B. Andersonand D. Ma, Lipids Health Dis. 8:33 (2009).

N-3 fatty acids have been associated with beneficial effects in patientswith cardiovascular disease and in reducing the risk of cardiovasculardisease in healthy people. Similar positive effects have been reportedin stroke. Accordingly, the American Heart Association, as well as otherhealth agencies, has issued recommendations for increased intake of n-3fatty acids in the diet. P. Kris-Etherton et al. Circulation106:2747-2757 (2002). Possible mechanisms for the observed effects ofn-3 fatty acids on cardiovascular health include hypotriglyceridemiceffects, hypotensive effects, reduction in platelet aggregation, andstabilizing effects on the myocardium itself.

The benefit of n-3 fatty acids in some conditions may be attributed tobroad anti-inflammatory effects. EPA and DHA give rise to resolvins,which are anti-inflammatory mediators with inflammation-resolving andimmunomodulatory functions. For example, EPA and DHA exhibit inhibitoryeffects on leukocyte chemotaxis and alter the production of inflammatorycytokines through reducing activation of NF-κB in immune cells. P.Calder, Int. Rev. Immunol. 28:506-534 (2009). In general, n-3 PUFAs areassociated with reduced pro-inflammatory T cell responses. When n-3fatty acids are increased in animal diets, the T cell membranemicrodomain composition in lipid rafts is altered, resulting indecreased NF-κB activation, IL-2 production, and cellular proliferation.Specifically, n-3 PUFAs affect the distribution and partitioning of theearliest signaling mediators of T-cell activation, such as proteinkinase C. Y. Fan et al., J. Immunol. 173:6151-6160 (2004). N-3 fattyacids have also been shown to reduce MHC class II expression ondendritic cells, effectively decreasing antigen presentation to T cells,whereas n-6 fatty acids are associated with increased antigenpresentation activity. Sanderson et al., J. Leukoc. Biol. 62:771-777(1997). In a monocyte cell line and in intraperitoneal macrophages, DHAand EPA have anti-inflammatory properties mediated through a Gprotein-coupled receptor 120 (GPR120). As a result, these fatty acidsdisplay antidiabetic effects in vivo via suppressing macrophage-inducedtissue inflammation. D. Oh et al., Cell 142(5):687-698 (2010). Thevarious immunomodulatory functions of n-3 PUFAs indicate they may beinfluential in many human diseases.

n-6 Fatty Acids

Like n-3 fatty acids, n-6 fatty acids, such as ARA, play a crucial rolein neural development and brain function, with ARA accumulationoccurring in the brain during pre- and post-natal development. B.Koletzo et al., J. Perinat. Med. 36(1):5-14 (2008). N-6 fatty acids aregenerally important for normal development and immunity, and alsostimulate skin and hair growth, maintain bone health, regulatemetabolism, and maintain the reproductive system.

Long-Chain PUFA Supplements

For over a decade, health agencies have recommended the consumption ofn-3 fatty acids in the diet due to their health benefits. DHA and EPAare commercially available as triglycerides or in esterified form innutritional supplements or prescription products (e.g., LOVAZA®,OMACOR®, and Vascepa™). DHA supplements may be derived from fish oil, orfrom vegetarian sources such as flaxseed oil or algae. Supplements maybe powder, liquid beverage, or tube-feeding formulas.

Infant formula is subject to the Federal Food, Drug, and Cosmetic Act,which defines infant formula as “a food which purports to be or isrepresented for special dietary use solely as a food for infants byreasons of its simulation of human milk or its suitability as a completeor partial substitute for human milk.” The FDA defines infants as peoplenot more than 12 months old. 21 CFR 105.3(e). The main n-3 fatty acid inhuman milk is DHA, averaging 7-8 mg/dL (ranging from 0.17% to 1.0% oftotal fatty acids). R. Yuhas et al., Lipids 41(9):851-858 (2006). Theamount of DHA in human milk is mostly a reflection of maternal DHAintake.

Commercially available TG-LCPUFA-supplemented infant formulas includeEnfamil formulas, such as Enfamil LIPIL® and Enfamil PREMIUM®, Baboo,Earth's Best Organic, Nestle formulas, such as Nestle Gerber GOOD START®and Nestle NAN®, Nutricia formulas such as NEOCATE® and APTAMIL®,Parent's Choice Organic, Pfizer's SMA GOLD®, Similac formulas, such asSimilac ADVANCE®, Similac EARLY SHIELD®, and ISOMIL®, and Ultra BrightBeginnings. Other infant formulas may also be supplemented withTG-LCPUFA. TG-LCPUFA-supplemented formulas may be milk-based orsoy-based, and may be organic. In the U.S., TG-LCPUFA-supplementedinfant formula accounts for approximately 90% of product sales (MeadJohnson Nutrition).

TG-LCPUFA may also be added to follow-on formulas and drinks fortoddlers, elderly, and other people needing nutritional support ordietary supplementation with long-chain fatty acids. Examples of such aproduct include ENSURE®, PEDIASURE®, CARNATION®, BOOST®, CERELAC®, andSOUVENAID®. In addition, specialized formulas that are supplemented withTG-LCPUFA or esters of LC-PUFAs may be used in connection with themethods and devices of the invention in patients requiring tube feeding.For example, enteral formulas are commonly used in pre-term infants,patients with renal failure, gastrointestinal diseases or conditionscausing impaired GI function, bowel resection, fat malabsorption,malnutrition, pancreatitis, hyperglycemia/diabetes, liver failure, acuteand chronic pulmonary disease, or an immunocompromised state. For areview of commercially available enteral formulas, see A. Malone, Pract.Gastr. 29(6):44-74 (2005). Nutritional formulas may be standard,elemental, or specialized based on a patient's disease or condition.Commonly used standard formulas include, for example, ISOCAL®, NUTREN1.0®, NUTREN 1.58, NUTREN 2.0®, OSOMLITE 1.0®, OSMOLITE 1.2®,FIBERSOURCE 1.2®, JEVITY 1.28, JEVITY 1.58, PROBALANCE®, ISOSOURCE 1.58,DELIVER 2.0®, NOVOSOURCE 2.0®, and TWOCAL HN®. Elemental formulas maycontain macronutrient sources including polymeric and hydrolyzedformulas and may be fiber enhanced. Disease specific formulas include,for example, renal formulas such as MAGNACAL RENAL®, NEPRO®, NOVASOURCERENAL®, SUPLENA®, and NUTRI-RENAL®.

Gastrointestinal (GI) formulas may used for the nutritional managementof patients with impaired GI function including in patients with severeprotein or fat malabsorption, extensive bowel resection, cysticfibrosis, cerebral palsy, short bowel syndrome, IBD, pancreatitis,Crohn's disease, diarrhea, GI fistula, Celiac disease, malabsorptionsyndromes, trauma/surgery, radiation enteritis, intestinal failure,chylothorax. These formulas are also used for early post-operativefeeding, trophic feeding, total parenteral nutrition (TPN) alternative,and dual feeding with TPN. GI formulas include, for example, PEPTAMEN®,which is made up of 70% medium-chain triglycerides to decrease thepotential for fat malabsorption and 30% long-chain triglycerides,VIVONEX PLUS®, and VIVONEX PEDIATRIC®.

Unfortunately, for people suffering from impaired ability to hydrolyzelong-chain triglycerides or esters of long-chain fatty acids, such as,e.g., those with compromised pancreatic output or those suffering frompancreatic insufficiency, even supplementing such formulas with DHA,EPA, and other n-3 fatty acids may not be enough to realize the benefitsassociated with these compounds. Long-chain triglycerides or fatty acidesters must be metabolized to monoglycerides and/or free fatty acids inorder to be properly absorbed in the gut. The invention provides methodsof utilizing existing commercially available long-chain PUFA supplementsor newly designed formulas supplemented with long-chain PUFAs to provideready to use formulas containing significantly higher concentrations oflong-chain monoglycerides and/or free fatty acids. In some embodiments,the methods will be particularly effective at providing long-chainmonoglycerides and/or free fatty acids produced from DHA, EPA, and ARAtriglycerides or esterified DHA, EPA, and ARA so that the formula willprovide the maximum benefit associated with these critical fatty acidsto people who otherwise would not be able to hydrolyze and absorb them.

Reduced Ability to Hydrolyze Long-Chain Triglycerides and Fatty AcidEsters

Pancreatic insufficiency is one of the conditions that leads to areduced ability to hydrolyze long-chain triglycerides. Pancreaticinsufficiency is characterized by insufficient production of exocrinepancreatic enzymes, including pancreatic lipase. Pancreaticinsufficiency may occur naturally during various stages of human life.For example, the secretion of pancreatic lipase begins at low levels ataround 30 weeks gestation and remains low during the first year of life.Therefore, infants, and especially pre-term infants, may experiencepancreatic insufficiency. As a result, if they are not breast feeding,these infants are susceptible to poor fatty acid hydrolysis andabsorption, and are deprived of the benefits associated with ingestionof DHA, EPA, and other LC-PUFAs.

On the other end of the spectrum, otherwise healthy elderly may alsoexperience pancreatic insufficiency or other reduced ability tohydrolyze LC-PUFA triglycerides or esterified LC-PUFAs due to changes inthe pancreas that occur as part of the natural aging process. Thesechanges may include atrophy, fibrosis, sclerosis, or lipomatosis of thepancreas. As a result, the elderly may experience symptoms ofmaldigestion including malnutrition, steatorrhoea, diarrhea, abdominalpain and weight loss because of reduced exocrine pancreatic enzymesecretion. K. Herzig et al., BMC Geriatrics 11:4-8 (2011).

Pancreatic insufficiency or other reduced ability to hydrolyze LC-PUFAtriglycerides or esterified LC-PUFAs may also result from disease ortrauma. For example, pancreatitis is a condition of inflammation in thepancreas which results in pancreatic insufficiency. Pancreatitis may beeither acute or chronic, and includes pancreatitis caused by alcoholism,idiopathic chronic pancreatitis, hereditary pancreatitis, traumaticpancreatitis, acute necrotizing pancreatitis, and autoimmunepancreatitis. Cystic fibrosis is also a cause pancreatic insufficiency,particularly in children and adolescents. Disorders that result in adecrease in intraduodenal pH, such as gastrinoma (Zollinger-Ellisonsyndrome), can inactivate lipase and cause pancreatic insufficiency.Pancreatic insufficiencies can also be caused by surgeries of thegastrointestinal tract in which portions of the stomach or pancreas areremoved, pancreatic cancer, gastrointestinal diseases such as stomachulcers, celiac disease, or Crohn's disease, or in autoimmune disorderssuch as systemic lupus erythematosus (SLE) or inflammatory bowel disease(IBD).

Other causes of a reduced ability to digest TG-LCPUFAs, esterifiedLC-PUFAs, and/or other long-chain triglycerides and fatty acid estersinclude, for example, irritable bowel syndrome, hypertriglyceridemia,malnutrition, including severe protein-calorie malnutrition, pancreaticand duodenal neoplasms, abdominal radiotherapy, hemochromatosis, primarysclerosing cholangitis, primary biliary cirrhosis, Shwachman's syndrome,trypsinogen deficiency, enterokinase deficiency, or an isolateddeficiency of lipase. D. Kasper et al., Harrison's Principles ofInternal Medicine 16^(th) Ed. (2004). A reduced ability to digestlong-chain triglycerides or esterified long-chain PUFAs may also resultfrom bowel resection, cystic fibrosis, cerebral palsy, short bowelsyndrome, IBD, pancreatitis, Crohn's disease, diarrhea, GI fistula,Celiac disease, malabsorption syndromes, trauma/surgery, particularly GItrauma or surgery, radiation enteritis, intestinal failure, chylothorax,cancer, particularly pancreatic or GI cancer, and/or wound healing.Although the exact cause is unknown, children with attention deficithyperactivity disorder (ADHD) have also reduced levels of LC-PUFAs.Burgress et al., Am. J. Clin. Nutri. 71(suppl):327S-30S (2000).

Cystic fibrosis (CF) patients, for example, have been shown to havereduced levels of LC-PUFAs. Peretti et al., Nutrition & Metabolism2:11-28 (2005). CF patients receiving pancreatic enzyme replacementtherapy frequently continue to suffer from fat malabsorption.Kalivianakis, American Journal of Clinical Nutrition 69:127-134 (1999).In some embodiments, the invention provides formulas and methods forimproving absorption of fats, such as, e.g., LC-PUFAs, in CF patients.In some embodiments, the invention provides formulas and methods forinducing weight gain in CF patients

While cachexia and weight loss are common in the advanced stages of manycancers due to the catabolic state of tissues, diversion of nutrients,and malabsorption in advanced stages, pancreatic cancer (PC) is unusualin that weight loss and malabsorption are present in 80%-90% of patientsat the time of diagnosis. Malabsorption from exocrine deficiency largelyaccounts for weight loss and is due to loss of pancreatic parenchyma,blockage of the pancreatic duct preventing enzymes from reaching thegut, and surgical procedures. The common end result of all thesemechanisms is steatorrhea and weight loss. Damerla et al., J of SupportOncology 6:393-396 (2008). Weight stabilization in PC is associated withimproved survival and quality of life. Davidson et al., ClinicalNutrition 23, 239-247 (2004). In some embodiments, the inventionprovides formulas and methods for improving absorption of fats, such as,e.g., LC-PUFAs, in PC patients. In some embodiments, the inventionprovides formulas and methods for inducing weight gain in PC patients.

Some embodiments of the invention improve upon current treatment optionsfor pancreatic insufficiency and other conditions that reduce theability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and/or otherlong-chain triglycerides and fatty acid esters. In a patient withreduced ability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and/orother long-chain triglycerides and fatty acid esters, merely increasingconsumption of these nutrients without improving hydrolysis can causesteatorrhea, abdominal pain, cramping, diarrhea, and othergastrointestinal complications. Pancreatic enzyme replacement therapycan also lead to complications. It has been observed that large amountsof pancreatic digestive enzymes can damage the large intestine resultingin fibrosing colonopathy. D. Bansi et al., Gut 46:283-285 (2000); D.Borowitz et al. J. Pediatr. 127:681-684 (1995). Another significantdanger posed by lipase supplements is allergic reaction, as manycommercial lipase supplements are derived from animal sources. Thus,embodiments of the invention that provide pre-hydrolyzed long-chaintriglycerides or long-chain PUFA esters, with or without added lipase,will provide better and safer methods for treating pancreaticinsufficiency or other reduced ability to digest long-chaintriglycerides or esterified long-chain PUFAs.

While both n-3 and n-6 fatty acids are important during development, n-3fatty acids are believed to be more critical than n-6 fatty acids laterin life. In some subjects, particularly some adults, it may be desirableto increase the ratio of (DHA and EPA):ARA. In particular, cysticfibrosis patients may benefit from increasing the ratio of (DHA andEPA):ARA in their plasma. Unfortunately, currently available adultformulas generally have a low ratio of n-3:n-6 fatty acids. Moreover, insubjects with impaired hydrolysis of TG-LCPUFAs, simply increasingconsumption of n-3 TG-LCPUFAs is unlikely to significantly improve the(DHA and EPA):ARA ratio in the subject, and the resulting increase inundigested TG-LCPUFAs could cause gastrointestinal problems.

Accordingly, some embodiments of the invention provide formulas andmethods for increasing the ratio of (DHA and EPA):ARA in a subject,particularly in an adult subject. For example, some embodiments providemethods of preparing an adult formula in which a formula comprising n-3triglycerides and/or esters is exposed to a lipase that hydrolyzes n-3triglycerides and/or esters. In some embodiments, the prepared formulacomprises a higher ratio of n-3:n-6 monoglycerides and/or free fattyacids, e.g., a higher ratio of free DHA and EPA to free ARA, than in thecorresponding formula without lipase treatment. In some embodiments, theformula comprises more n-3 monoglycerides and/or free fatty acids thann-6 monoglycerides and/or free fatty acids, e.g., more free DHA and EPAthan free ARA. In some embodiments, the formula is prepared by exposingit to a lipase that has higher activity toward n-3 triglycerides and/oresters than n-6 triglycerides and/or esters. In some embodiments, theenzyme is RO enzyme. The invention also provides a formula in which theratio of n-3:n-6 free fatty acids and/or monoglycerides is higher thanthe ratio of n-3:n-6 fatty acids found in the subject's plasma, e.g., aformula in which the ratio of free DHA and EPA to free ARA is higherthan in the subject's plasma. The invention also provide methods inwhich such a formula is administered to an adult subject. In someembodiments, the subject has cystic fibrosis.

Reduced Ability to Hydrolyze Long-Chain Fatty Acids in Pre-Term Infants

Long-chain PUFAs are critical in infants for normal nervous system andretinal development and are highly accumulated in the cell membranes ofthe brain and retina starting at 30 weeks gestation. C. Martin et al.,J. Pediatr. 159(5):743-749 (2011); A. Lapillone et al., Leukotrines Ess.Fatty Acids 81:143-150 (2009); J. McCann et al., Am. J. Clin. Nutr.82:281-295 (2005); M. Martinez et al., J. Pediatr. 120:S129-S138 (1992).Normally fatty acids, including DHA, EPA, and ARA, as well as thelipases needed to break these fatty acids down to monoglycerides andfree fatty acids are provided to the fetus through the placenta and thento infants through breast milk. Pre-term infants are at a significantlyhigher risk for an inadequate supply of fatty acids due to the shortenedgestation time followed by their dependence on external sources forfatty acids after birth. C. Martin et al., J. Pediatr. 159(5):743-749(2011). Additionally, because pre-term infants do not produce sufficientlevels of pancreatic lipase, they consequently have difficultyhydrolyzing any long-chain fatty acids that are provided in theirformula.

It has been demonstrated that pre-mature infants have less DHA and lowerDHA/ARA ratios in both the brain and retina compared to a full-terminfant. M. Martinez et al., J. Pediatr. 120:S129-S138 (1992).Additionally, in a retrospective study of the fatty acid profile ofpre-term infants, inadequate levels of long-chain PUFAs were associatedwith an increase of chronic lung disease and sepsis, possibly due todysregulated immune response. C. Martin et al., J. Pediatr.159(5):743-749 (2011). These studies and others suggest that, even withformulas supplemented with DHA and other long-chain triglycerides orlong-chain fatty acid esters, ensuring adequate levels of long-chainPUFAs in pre-term infants is a significant and potentially unmet need.The formulas, methods, and devices of the invention will allow pre-terminfants to receive sufficient amounts of long-chain fatty acids torecognize the associated medical benefits.

Reduced Ability to Hydrolyze Long-Chain Fatty Acids in Formula-FedInfants

Infants fed formula that have not been supplemented with fatty acids mayalso experience deficits in long-chain PUFAs. The levels of long-chainPUFAs were found to decline in infants fed unsupplemented formulacompared to infants fed breast milk. B. Koletzo et al., J. Perinat. Med.36(1):5-14 (2008). Even infants fed breast milk can experience deficitsin n-3 fatty acids as the amount of DHA in breast milk varies and iscorrelated with maternal dietary intake. A positive correlation betweenthe amount of DHA in breast milk and visual and language development inbreast-fed infants has been described. S. Innis, J. Pediatr. 143:S1-S8(2003). Thus, a diet containing DHA is recommended for women that arebreast-feeding. For formula-fed infants, all major formula manufacturershave introduced premium infant formulas with fats containing DHA andARA. However, reports on the benefits of those DHA and ARA enrichedformulas have been mixed. Some studies have shown significant advantagesin cognitive development when infants received long-chain PUFAcontaining formula, while others have not. B. Koletzo et al., J.Perinat. Med. 36(1):5-14 (2008); E. Sarkadi-Nagy et al., J. Lipid Res.45:71-80 (2004). Recently, infants fed Enfamil LIPIL® containing DHA andARA during the first year of life experienced improved immune outcomes,including improved respiratory health, compared to infants fed the sameformula without lipids. E. Birch et al., J. Pediatr. 156(6):902-906(2010). Overall, however, the pre-clinical data to date has not shownconsistent benefits of current long-chain PUFAs-supplemented formulasfor infant development.

One explanation for the inconsistent results in these studies is thatsome infants are not able to absorb the necessary amount of criticalfatty acids through the gut even when ingesting formula supplementedwith long-chain triglycerides or long-chain fatty acid esters. Thisinability to absorb fatty acids may be due to the infants' low levels ofendogenous pancreatic lipase. Because lipases are typically transferredto an infant through breast milk, formula-fed infants may not havesufficient levels of lipase to break down the long-chain PUFAs or PUFAesters to monoglycerides and/or free fatty acids, for absorption by thegut. As a result, infants fed LC-PUFA-supplemented formula still haveless absorption of LC-PUFAs compared to infants fed breast milk. Onceagain, there is a clear need to not simply provide fatty acidsupplements, but to enable hydrolysis and absorption of these fattyacids.

Adding lipase to government regulated infant formulas (or e.g., medicalnutritional formulas) could require significant development work toscreen, stabilize and formulate a suitable lipase supplement. Inunregulated formulas, without sufficient testing, issues involvinglipase stability, lack of specificity, purity, and/or interference withother materials may result in the use of excess or potentially harmfullevels of enzyme. Adding copious amounts of a new substance, beyondregulatory hurdles, also introduces another variable that could affecthow well a person with reduced ability to hydrolyze long-chaintriglycerides, particularly an infant, will tolerate a formula. Thisproblem persists in formulas described, e.g., in U.S. Pat. No. 5,902,617(Pabst) and U.S. Pat. No. 4,944,944 (Tang).

Embodiments of the invention solve these various problems by providing anutritional formula that, as-fed, provides increased amounts ofessential monoglycerides and free fatty acids that may be readilyabsorbed through the gut of an infant. As a result, formula-fed subjectscan be provided with the benefits of DHA, EPA, and ARA. In someembodiments, the nutritional formula introduces no new ingredientsexcept pre-hydrolyzed fats that are present in existing formulas. Incertain embodiments, formula-fed babies are provided the fatty acidbenefits obtained by breast-fed infants, without exposure to lipasesupplements. In other embodiments, nutritional formula of the inventioncontains a highly specific lipase that allows for the use of a minimalamount of lipase added to infant formula to provide increased amounts oflong-chain monoglycerides and free fatty acids, particularly DHA, EPA,and ARA.

In some embodiments, the nutritional formula leads to improvedabsorption of fatty acids. In some embodiments, a subject ingests thenutritional formula for 3 days, 5 days, 7, days, 10 days, 14 days, 30days, 60 days, or more. In some embodiments, such ingestion of anutritional formula of the invention reduces the total fat in the stool,and specifically can reduce the levels of DHA, ARA, and/or EPA in thestool. In some embodiments, this reduction is measured relative to thesubject's stool composition prior to beginning to ingest the nutritionalformula. In some embodiments, this reduction is measured relative to thestool composition of a subject fed a nutritional formula that has notbeen exposed to lipase prior to ingestion, such as a currently availablenutritional formula. The levels of total fat, DHA, ARA, and/or EPA inthe stool may be reduced by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or more. In certain embodiments, the levels of total fat,DHA, ARA, and/or EPA in the stool are reduced by between 50 and 80%. Insome embodiments, the level of total fat in the stool is reduced by atleast 50%. In some embodiments, the level of at least one LC-PUFA (suchas DHA, ARA, or EPA) in the stool is reduced by at least 50%. In someembodiments, the level of at least one LC-PUFA (such as DHA, ARA, orEPA) in the stool is reduced by at least 60%. In some embodiments, thelevels of DHA, ARA, and EPA in the stool are each reduced by at least50%. In some embodiments, the levels of DHA, ARA, and EPA in the stoolare each reduced by at least 60%. In some embodiments, ingestion of thenutritional formula improves plasma, erythrocyte, and tissue accretionof fat levels, including levels of DHA and ARA. Tissues may includeretina, heart, adipose, and kidney tissue. In some embodiments,ingestion of the nutritional formula increases the level of DHA, ARA, orboth in the plasma, erythrocytes, or both. In some embodiments,ingestion of the nutritional formula increases the level of DHA, ARA, orboth in the retina. In some embodiments, ingestion of the nutritionalformula increases the level of DHA, ARA, or both in the heart. In someembodiments, ingestion of the nutritional formula increases the plasmalevel of triglycerides, cholesterol, HDL, and/or LDL. In someembodiments, ingestion of the nutritional formula increases the ratio ofHDL to LDL in the subject's plasma.

In some embodiments, ingestion of the nutritional formula increases theplasma level of vitamin A and/or vitamin E. Without intending to bebound by theory, it is believed that this increase is due to the factthat vitamins A and E are typically provided as esters, which must behydrolyzed. Exposure to lipase in various methods and compositions ofthe invention is believed to improve hydrolysis of these vitamin esters,leading to greater accumulation of vitamins A and E in the plasma.

In some embodiments, ingestion of the nutritional formula has beneficialeffects without significantly increased accumulation of fat in theliver. Fatty liver disease (FLD) is characterized by increasedaccumulation of fat, especially triglycerides, in the liver cells. Thecondition is also associated with other diseases that influence fatmetabolism. It is normal for the liver to contain some fat and byitself, this causes no symptoms. In some patients, fatty liver may beaccompanied by hepatic inflammation and liver cell death(steatohepatitis). There is also an association with liver cancer(hepatocellular carcinoma). Insulin resistance, as well as increasedconsumption of carbohydrates and saturated fatty acids, and a low intakeof fiber and omega-3 fatty acids, are all positively associated with thepathogenesis of FLD.

Causes of FLD include diet, medications, diseases, and medicalconditions. Consumption of excess calories can cause FLD; the excesscaloric intake overwhelms the liver's ability to metabolize fat in anormal fashion, which results in fat accumulation in the liver. A numberof medications, including as tamoxifen, amiodarone injection, amiodaroneoral, and methotrexate are associated with FLD. Fatty liver is alsoassociated with type II diabetes, obesity, and high triglyceride levelsin the blood, celiac disease, and Wilson's disease (abnormality ofcopper metabolism), rapid weight loss, and malnutrition.

Lipase

Pancreatic insufficiency and other conditions associated with reducedability to hydrolyze long-chain triglycerides or long-chain fatty acidesters is currently treated with supplementary digestive enzymes,including pancreatic lipase. However, pancreatic enzymes, andparticularly pancreatic lipase present in these supplements, are oftensensitive to degradation by gastric acid and pepsin so that only a smallfraction of the ingested enzymes reach the duodenum in active form. E.Ville et al., Digestion 65:73-81 (2001). Unfortunately, many of the acidprotective coatings have potential safety concerns for infantpopulations or immune compromised patients since a significant portionof the delivered weight is the plastic coating. Moreover, although acidprotective coatings have helped, some degree of malabsorption persists,causing patients with pancreatic insufficiency to require increasingdoses of enzyme supplements. This persistence of fatty acidmalabsorption of enterally coated enzymes may be due to the fact thatthe duodenum and upper jejunum in patients with pancreatic insufficiencyare often acidic environments, so that the expected raise in pH is notachieved and the protective coating is not properly dissolved to releasethe enzyme. D. Graham, New England J. Med. 296(23):1314-1317 (1977).Both of these problems have been addressed by increasing the dose oflipase administered. Unfortunately, as previously noted, high doses ofpancreatic enzyme supplement have been found to be associated withfibrosing colonopathy. Thus, some embodiments of the invention providenutritional formulas that comprise higher percentages of long-chainmonoglycerides and/or free fatty acids without containing added lipase.Some embodiments provide nutritional formulas that comprise an optimizeddose of lipase, as described herein.

Lipases can be obtained from animal, plant, and many natural orgenetically engineered microorganisms. Many, if not most, commerciallyavailable dietary lipase supplements are derived from animals and areparticularly susceptible to degradation by digestive enzymes. A lessfrequently used alternative is microbial lipase, i.e., lipase producedin bacteria or fungus, such as, e.g., yeast. Microbial lipases retainactivity over a wider pH range than animal or plant lipases, thuseliminating the need for enteric coated tablets. However, microbialenzymes tend to be degraded by trypsin in the small intestine, therebyreducing their availability to breakdown triglycerides and esters in thegut. In certain embodiments, the lipase used in the formulas, methods,or devices of the invention are bacterial lipases, fungal lipases, orboth.

The specificity and kinetics of individual lipases can varysignificantly. Specificity of lipases is controlled by the molecularproperties of the enzyme, structure of the substrate and factorsaffecting binding of the enzyme to the substrate. Types of specificityinclude substrate specificity, i.e., a given lipase may be more activein breaking down a type of fatty acid than another lipase, andpositional specificity, which involves preferential hydrolysis of esterbonds in positions 1 and/or 3 of the glycerol backbone of atriglyceride.

It has now been determined that lipase produced by Chromobacteriumviscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopusoryzae have greater specificity for DHA, EPA, and ARA than otherlipases, such as lipase produced by Candida rugosa, Rhizomucor miehei,Penicilium camemberti, Aspergillus niger, and Aspergilis oryzae. As aresult, lipase supplements, or lipase supplemented nutritional productscomprising Chromobacterium viscosum, Pseudomonas fluorescens,Burcholderia cepacia, and/or Rhizopus oryzae will provide increasedhydrolysis of TG-DHA, TG-EPA, and/or TG-ARA. Accordingly, one aspect ofthe invention provides lipase supplements or lipase supplementednutritional products comprising Chromobacterium viscosum lipase,Pseudomonas fluorescens lipase, Burcholderia cepacia lipase, and/orRhizopus oryzae lipase. In some embodiments, the lipase isChromobacterium viscosum lipase, Pseudomonas fluorescens lipase, orRhizopus oryzae lipase. In certain embodiments, the lipase is Rhizopusoryzae lipase.

Reference to the lipase of certain species, such as Chromobacteriumviscosum lipase, Pseudomonas fluorescens lipase, Burcholderia cepacialipase, and Rhizopus oryzae lipase, does not necessarily mean that thelipase was prepared directly from the native host species. For example,the same lipase could be produced recombinantly in another host cell.

Another aspect of the invention is a method of increasing the absorptionof DHA, EPA, and/or ARA by administering one or more of Chromobacteriumviscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopusoryzae lipases, as a dietary supplement, or by pre-hydrolyzing a formulacontaining DHA, EPA, and/or ARA with one or more of these enzymes. Insome embodiments, the lipase is Chromobacterium viscosum lipase,Pseudomonas fluorescens lipase, or Rhizopus oryzae lipase. An additionalaspect of the invention provides lipases with specific activities forDHA, EPA, and/or ARA that are comparable to the specific activities ofone or more of Chromobacterium viscosum, Pseudomonas fluorescens,Burcholderia cepacia, and Rhizopus oryzae as determined by reverse-phasehigh performance liquid chromatography (RP-HPLC) and described inExample 1. In some embodiments, the lipase has specific activities forDHA, EPA, and/or ARA that are comparable to the specific activities ofone or more of Chromobacterium viscosum lipase, Pseudomonas fluorescenslipase, or Rhizopus oryzae lipase. One embodiment of the invention is anutritional formula that contains less than 5,000 units of lipase (withunits assessed in a standard olive oil assay, such as described inPharmaceutical Enzymes: Properties and Assay Methods, R. Ruyssen and A.Lauwers (Eds) Scientific Publising Company, Ghent, Gelgium (1978)). Inother embodiments, the nutritional formula contains less than 3,000units of lipase. In some embodiments, the nutritional formula containsless than 1,000 units. In certain embodiments, the formula containingless than 5,000, less than 3,000, or less than 1,000 units of lipase isan infant formula or a medical nutritional formula.

Immobilized Lipase

Processes for immobilizing enzymes and other proteins to insolublesupports are well-known and described in the literature. Immobilizationof lipase may improve the stability of the enzyme, render it reusable,and allow products to be readily separated from the enzyme withoutcontamination by lipase. In some embodiments, the lipase is covalentlybound to a solid support, however, non-covalent binding may also beused. Suitable methods of immobilization of lipase include, for example,adsorption, ionic binding, covalent binding, cross-linking,encapsulation, and entrapment onto hydrophobic or hydrophilic polymericand inorganic matrices. See Y. Ren et al., BMC Biotechnol. 11:63 (2011);V. R. Murty et al., Biotechnol. Bioprocess Eng. 7:57-66 (2002). Lipasemay be immobilized by binding directly to a support material or througha linker. See, e.g., Stark and Holmberg, Biotechnol. and Bioeng.34(7):942-950 (1989).

Immobilization by adsorption is reversible and typically involveshydrophobic forces. It is simple and inexpensive, but has thedisadvantage of incomplete immobilization or leaking enzyme from theinsoluble support. Examples of immobilized lipase using this method canbe found in E. Lie et al., Chem. Technol. and Biotechnol. 50:549-553(1991) (Candida cylindracea lipase, zeolite support); M. Basri et al.,J. Chem. Technol. and Biotechnol. 59:37-44 (1994) (Candida rugosalipase; polymer support); H. Gunnlaughsdottir et al., Enzyme andMicrobiol. Tech. 22:360-367 (1998) (Humicola lanuginose lipase; glassbeads support). Supports suitable for immobilization by adsorptioninclude, e.g., ceramic beads such as Toyonite (Toyo Denka Kogyo Co.,Ltd.).

Ionic binding is based on electrostatic interactions between the lipaseand differently charged ionic groups on matrices such as e.g.,DEAE-cellulose or DEAE-Sephadex on a solid support. Ionic binding causesminimal change to the conformation of the lipase and yields immobilizedlipase with high activity in most cases. It should be kept in mind,however, that although the binding force between the enzyme and thesupport is stronger than when using adsorption, it is not as strong ascovalent binding and thus, leaking of lipase from the support may occur.

Covalent binding is based on covalent bonds between a support materialand a functional group on an amino acid on the surface of the lipase.The functional groups that may take place in this binding of enzyme tosupport can be amino, carboxyl, sulfhydryl, hydroxyl, imidazole, orphenolic groups which are not essential for the catalytic activity ofthe lipase. In order to protect the active site, immobilization can becarried out in the presence of substrate or a competitive inhibitor. Asignificant advantage to using covalent binding of lipase to a supportmaterial is the strength of the bond, i.e., the stability of theimmobilization. For an example of lipase immobilized by covalentbinding, see S. Emi et al., European Polymer Journal 30(5):589-595(1994). Supports suitable for covalent binding include, e.g., Immobead™(ChiralVision).

Cross-linking involves joining the lipase to itself to form athree-dimensional structure or joining the lipase to a solid structureusing a crosslinking agent. For example, lipase may be cross-linked tochitosan beads. See S. H. Chiou et al., Prep. Biochem. Biotechnol.37(3):265-275 (2007). Immobilization of lipase by encapsulation usuallyinvolves the formation of a porous coating or semi-permeable membranearound the lipase so that the lipase is contained inside the porousmaterial, but triglycerides and esters may pass freely through.Immobilization of lipase by entrapment involves restricting the movementof the enzyme by trapping it in a lattice structure. Alginate beads maybe used for this type of immobilization. I. Bushan et al., J. Bioactiveand Compatible Polymers 23(6):552-562 (2008). Synthetic and naturalpolymers may also be used. See also, G. Fernandez-Lorente et al., J. Am.Oil Chem. Soc. (published online 14 Dec. 2010) and G. Fernandez-Lorenteet al., J. Am. Oil Chem. Soc. 88:1173-1178 (2011)

In certain embodiments, the formulas, methods, and devices of theinvention will utilize lipase that has been crystallized andcross-linked for increased stability as described in U.S. Pat. No.6,541,606 (Margolin), either with or without another form ofimmobilization, such as encapsulation.

In some embodiments, lipase is immobilized to magnetic nanoparticles(MNPs). These MNPs may be coated by linkers or polymers containing aminoor epoxy functional groups to which the lipases are reacted. Onesuitable coating for MNPs is, e.g., polydopamine. See, e.g., Y. Ren etal., BMC Biotechnology 11:63 (2011). The use of MNPs for lipaseimmobilization has advantages such as biocompatibility, supermagnetism,small size, and low toxicity. The magnetic properties of thenanoparticles facilitate removal of the lipase from solution and alsoprovides another means for attaching the MNP-lipase to a solid support.

In some embodiments, the immobilized lipase is a microbial lipase. Insome embodiments, the immobilized lipase is selected from bacteriallipases. In some embodiments, the immobilized lipase is one or morelipases selected from Chromobacterium viscosum, Pseudomonas fluorescens,Burcholderia cepacia, and Rhizopus oryzae.

In certain embodiments, the lipase (whether immobilized or not) is addedto formula for 1, 2, 3, 4, 5, 10, 20, 30 minutes or more. The hydrolysisof LC-PUFA triglycerides and esters is measured by RP-HPLC. In certainembodiments, the percent hydrolysis of LC-PUFA triglycerides and estersis 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% by 30 minutes.In embodiments, the percent hydrolysis of LC-PUFA triglycerides andesters is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% by 20minutes. In embodiments, the percent hydrolysis of LC-PUFA triglyceridesand esters is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% by 10minutes. In certain embodiments, the lipase is Rhizopus oryzae lipase.

Devices Comprising Immobilized Lipase

According to various embodiments, the present disclosure providesdevices and methods for preparing nutritional products. The devices andmethods can be used to expose infant formula or other nutritionalproducts to lipases prior to consumption. The lipases will accordinglybreakdown fats and oils with subsequent release of free fatty acids andmonoglycerides. The devices and methods will allow convenient means forpreparing formula or other nutritional products. In some embodiments,the devices and methods allow infants or others who consume the productsto avoid consuming exogenous lipase. In some embodiments, the devicesand methods allow for production of formulas that contain monoglyceridesand/or free fatty acids but do not contain any significant amount oflipase (as determined by ELISA).

FIGS. 1, 2A-2C, 3A-3C, 4A-4B, 5A-5B, 9A-9C, 10A-10C, 11A-11C, 12,13A-13B, 14, 15, 16, 17A-17B, 18, 19, 20, and 21 illustrate devicesaccording to various embodiments of the present disclosure. As shown inFIG. 1, devices 100 of the present disclosure can include a container110 configured to hold infant formula 120 or other liquid nutritionalproducts. As described in detail below, the container 110 can includelipases that are immobilized such that formula 120 that is fed to theinfant through a nasogastric tube 114 or other feeding mechanism (e.g.,a baby bottle) does not contain lipases in any appreciable amount. Forexample, the lipases can be immobilized on or in structures 150 foundalong the wall or otherwise within the container such that the lipasesare in fluid contact with formula 120 within the container. Further, asis discussed with reference to various embodiments below, formula can beadded to the container 110, in various ways to allow enzymatic treatmentof lipases within the container 110. For example, fluid can be fedthrough a tube 112 or poured into the container, and can be subsequentlypassed through a nasogastric tube or other device for feeding.

Throughout this disclosure, the devices and methods will be referred tofor use in treating or preparing nutritional formula, such as, e.g.,infant formula and medical nutritional formula. It will be appreciatedthat the devices and methods can be used to treat or prepare any type ofnutritional formula for which it may be beneficial to provide lipasetreatment prior to consumption. Such products can include anynutritional formula to be consumed by someone with pancreaticinsufficiency or other reduced ability to hydrolyze long-chaintriglycerides or esterified long-chain PUFAs.

FIGS. 2A-2C and 3A-3C illustrate more detailed devices, according tovarious embodiments. As shown, the devices 200-202, 300-302 can includea container 210, 310 for holding liquid formula. The container 210, 310can include a variety of different types and configurations. Forexample, the container 210, 310 can include a glass or plastic jar orvial, a bag (e.g., silicone or other flexible material like an IV salinebag), a cylindrical container such as a syringe barrel, or othercontainer that is sized and shaped to hold a desired amount of formulaor other product.

As noted, the devices of the present disclosure can allow formula to beexposed to lipases to obtain the desired enzymatic effects, whileallowing the formula to then be conveniently consumed without consuminglipases. Accordingly, in various embodiments, lipases are immobilizedwithin the container 210, 310 such that, when the formula is removed(e.g., through a nasogastric tube, nipple for a baby bottle, or bytransferring the formula to another container), the lipases remain inthe container 210, 310 or can be removed from the formula prior toconsumption. In other embodiments, lipases are immobilized within thecontainer 210, 310, e.g., on removable solid supports, such that thelipases can be easily removed from the container, while leaving theformula in the container for later consumption.

FIGS. 2A-2C show one configuration for the container 210, along withcertain embodiments for immobilizing lipases within the container 210.As noted, the container 210 can include a variety of differentmaterials, sizes and shapes. In addition, the container 210 can includeone or more access ports 220, 230 for controlling flow of formula 260into and out of the container.

Lipases can be immobilized within the container 210 in a variety ofways. For example, lipases can be immobilized or contained withinstructures 250, 252 located inside the container 210 (FIGS. 2A and 2C).Additionally, or alternatively, lipases 251 can be immobilized on orcontained within the wall of the container 210 (FIG. 2B). Accordingly,as formula 260 is placed inside the container, the formula 260 comesinto contact with the lipases to produce the desired enzymatic effects.

As noted, lipases can be immobilized within the container by binding thelipases to structures 250, 252 within the container and/or to walls 251of the container. The structures within the container can have a varietyof configurations. For example, in certain embodiments, the structurescan include beads, balls, or any other structure that may themselves bemobile within the container such that the structures flow within theformula. For example, as shown in FIG. 4A, the structures 250 caninclude beads or balls having a surface wall 256 to which lipases 257can be bond. Further, it will be understood that the structures 250, 252can have a variety of different shapes or configurations (e.g., cuboid,ovoid, rod-like).

The structures 250, 252 and/or configuration of the wall of container210 can be configured to provide a desired surface area such thatformula is able to come in contact with a sufficient amount of lipaseduring an acceptable time period. For example, the structures 250, 252can include numerous beads 250 (FIG. 2A) or rod-like structures 252(FIG. 2C) to provide a high surface area for binding a sufficient amountof lipase. Alternatively, if longer time periods are available toincubate the formula with the lipases before consumption and/or lipaseswith high enzymatic activity are used, smaller amounts of lipase may besuitable.

In various embodiments, the structures 250, 252 and/or container areconstructed such that, as formula 260 is removed from the container forconsumption or storage, the lipase is not kept within the formula 260.For example, the beads 250 or rod-like structures can be sized such thatthey will not pass through a relatively small access port 230.Alternatively, or additionally, the structures can be attached to thecontainer wall and/or the container can include a screen or filter thatis sized to prevent movement of the structures with the formula 260.Further, the structures 250, 252 can have other properties thatfacilitate their separation from formula. For example, the structures250 can be formed of magnetic beads that can be removed by binding to amagnetic filter.

In some embodiments, rather than immobilizing lipases by attachment tostructures within the container 210 and/or container wall, the lipases257′ are contained within the structures 250, 252 and/or the containerwall 251. FIG. 4B illustrates one such an embodiment. As shown, thestructures 250′ can include beads or other shapes having a wall 256′.The wall may be formed of a semi-permeable materials that allows ingressand egress of formula 260 but not lipases 257′. Such encapsulation maysimilarly be used for other structures (e.g., 252) and/or for thecontainer wall such that the surface of the wall has a semi-permeablematerial, within which lipases may be contained.

The container may similarly have surface configurations that provide forincreased amounts of lipase, and/or increased contact of lipases withthe formula 260. For example, the wall of the container may have ridgesor other surface modifications to increase the surface area. Further,rather than including a single open space, the container may includevariations in the flow path, e.g., a long winding path to allowprolonged or longer exposure to lipases and/or a collection of channelsor tubes to which lipases are immobilized and through which formula mayflow. See, e.g., FIG. 1, element 150 and FIG. 21, element 2101.

In certain embodiments, the container may be manufactured andprepackaged with lipases in any of the embodiments described herein.During use, the container may be opened, and formula may be placed intothe container to contact the lipases for a sufficient time to producethe desired enzymatic effects. In other embodiments, structures such asbeads 250 or rod-like structures have lipases immobilized to theirsurfaces or contained/encapsulated within can be packaged anddistributed, and those structures can be placed into a separatecontainer containing formula. In some embodiments, it may be beneficialto shake or agitate the container comprising immobilized lipase andformula for a period of time.

As noted, formula 260 can be placed into the container 210 via variousaccess ports. For example, the container can include a top access port220 and/or a bottom access port 230. The ports 220, 230 can be used foringress and egress of formula respectively. In addition, a single portcan be used, or multiple ports may be used. The ports can comprise astructure configured to engage other devices that may be used forfeeding or transfer of fluids. For example, the ports can include aconnector such as a luer-lock connection, threads, and/or a conduit ortube that can engage a nasogastric tube. In addition, the ports can beconfigured to engage a baby bottle, baby bottle nipple, or any otherstructure to facilitate transfer of fluid to another container or toassist in feeding. Further, one or both ports 220, 230 can include avalve 140 (FIG. 1), 240 (FIG. 2A) or other fluid flow control mechanism.

FIGS. 3A-3C illustrate devices of the present disclosure, according tocertain embodiments. As shown, the devices 300-302 include a container310 for receiving formula 260. In addition, the container 310 caninclude a cap 322 or other closure device, such as a threaded top for ajar or bottle. Similar to the embodiments shown in FIGS. 2A-2C, thedevices can include structures 350, 351, 351′, and 353 that includelipases immobilized to their surfaces and/or encapsulated therein.

The embodiments of FIGS. 3A-3C can provide for more rapid separation offormula from the structures containing lipases. For example, as shown inFIG. 3A, the rod-like structures 350 can contain lipases, and afterenzymatic treatment of formula, the cap 322 can be removed tosimultaneously remove the structures 350 and lipases. Further, the cap322 can be replaced with another cap, bottle nipple, or other fluidconnection. Similarly, structures having other configurations, like theballs or beads 351, 351′ (FIG. 3B), can be sized for easy removal fromthe formula 260. For example, as shown, beads 351, 351′ are sized suchthat they can be easily removed manually or by filtration. Further, thecontainer 310 can provide lipases that are immobilized to or containedwithin its inner surface 353, and after enzymatic treatment, formula 260can be transferred to another container, or consumed by replacing thecap 322 with a bottle nipple or other connection to a feeding system.

Alternatively, or additionally, the structures 350, 351, 351′ can have apermeable outer wall with additional components providing immobilizedlipases contained therein. For example, structure 351′ (FIG. 3B),illustrates one embodiment wherein the structure 351′ has a permeableouter wall enclosing numbers beads 250. The outer wall can include amesh or other configuration that allows for easy movement of formulainto and out of the structure 351′ to provide contact with the beads250. Further, the beads can provide lipases, which can be immobilized totheir surfaces or encapsulated therein, as described in variousembodiments above.

As noted above, the structures containing lipases can be manufacturedand distributed as prepackaged components along with the container 310.Alternatively, or additionally, the structures can be packaged anddistributed separately from the container. For example, a cap 322containing rod-like structures 350 or beads 351, 351′, or otherwisehaving lipase contained therein or immobilized to it, can bemanufactured and distributed. The cap may be configured for connectionwith standard baby bottles, water bottles, or other container or devicethat may contain formula.

In other embodiments, lipases may be provided such that the lipasescontact formula as the formula is placed into a container and/or duringfeeding or removal from a container. For example, FIG. 5A illustratesone device 500, according to exemplary embodiments. The device 500 caninclude a standard bottle nipple, and lipases can be immobilized to aninner surface 510 of the nipple rim or nipple itself. As such, formulawill come into contact with the lipases during normal use. Similarly,lipases can be contained in or on other structures that may be used forfeeding, such as a fluid tube of a nasogastric feeding device.

Alternatively, the lipases can be provided in a separate elementconfigured to allow contact of formula with the lipases during normalfluid flow. For example, in one embodiment, the lipases can be containedwithin a housing 520 configured for engagement with a bottle closuresuch as a nipple (FIG. 5A) or a bottle cap/top (FIG. 5B). The housing520 can include a permeable walls that allows formula to flow throughits volume and contact lipases provided therein.

The lipases contained within the housing 520 can be provided in variousforms. For example, in some embodiments, the lipases are immobilized onbeads 550 within the housing 520 by bonding or encapsulation, asdescribed previously. Further, the housing 520 can include an open meshor other configuration that allows formula to flow through it. Forexample, with the bottle configuration shown in FIG. 5A, an open mesh orflow path through housing 520 will allow formula to contact lipases asthe formula exits a bottle during feeding. Alternatively, as shown inFIG. 5B, formula can be poured into or out of the top 530 of the housingto allow contact of formula with lipases during filling or emptying ofthe container 310. The top 530 and/or any other portion of the housing520 can be formed of a variety of materials. For example, the housing520 can be formed of a membrane that allows controlled fluid flow.Further, the top 530 may be formed of a semi-permeable membrane thatallows flow of liquid (formula) therethrough, but does not allow passageof lipases. Accordingly, the membrane forming the top 530 can serve toimmobilize the lipases within the container 310 without otherwisebinding or immobilizing the lipases within the container 310.

In various embodiments, the devices described above can includemodifications to improve or otherwise control lipase activity. Forexample, the containers 110, 210, 310 can include stirring systems toallow continuous movement of formula during an incubation period,thereby allowing the lipases to come into contact with fatty acids foundthroughout the fluid volume. Further, the devices can include systems tocontrol temperature to improve or control lipase activity.

Certain embodiments of the invention provide a container containingnutritional formula and a lipase. In some embodiments, the lipase is incontact with the nutritional formula in the container. In otherembodiments, the lipase and the nutritional formula are not in contactin the container. In some embodiments, the nutritional formula and thelipase are contained in separate compartments within the container. Insome embodiments, the nutritional formula is in dry form. In someembodiments, the nutritional formula is in liquid form. In someembodiments, the lipase is brought into contact with the nutritionalformula by releasing the lipase into the compartment containing thenutritional formula. In some embodiments, the lipase is brought intocontact with the nutritional formula by transferring the lipase and thenutritional formula into another container (e.g., by emptying the lipasecompartment and the nutritional formula compartment into the othercontainer). In some embodiments, liquid is added to the other containerbefore or after transferring the lipase and the nutritional formula intothe other container.

The devices according to the present disclosure can have a number ofdifferent shapes and/or configurations. For example, FIGS. 9A-9C,10A-100, 11A-110, 12, 13A-13B, 14, 15, 16, 17A-17B, 18, 19, 20, 21illustrate various additional shapes and configurations. In each of theconfigurations described with respect to those figures, lipases may beimmobilized using any of the methods described above (e.g., byimmobilizing lipases on structures such as beads within a device, and/orby immobilizing lipases within or on a wall or other surface of adevice). Further, the specific configuration may be selected to providea variety of different features, such as surface area, volume, amount oflipases, and/or exposure time of materials to enzymes.

The devices illustrated in FIGS. 9A-9C, 10A-10C, 11A-110, 12, 13A-13B,14, 15, 16, 17A-17B, 18, 19, 20, 21 can be configured to allow contactof lipases in a variety of ways. For example, in various embodiments, aportion or all of the device can be inserted within a container thatincludes formula in order to allow contact between the formula andlipases. In other embodiments, the device is configured for in-linetreatment of formula.

FIGS. 9A-9C illustrate various configurations for devices that may beplaced within a container to treat formula. As shown, the device 900,920 (FIGS. 9A and 9C) can have a variety of different shapes formed ofan outer wall 901, 901″ that encloses lipases. As indicated above, thelipases 902 can be immobilized in a variety of different ways, includingby attachment to beads. Furthermore, the devices 910 (FIG. 9B) caninclude more than one pocket or opening 903 formed in one or more walls901′. The specific configuration, number of pockets or openings, as wellas amount of lipase and/or volume of the device may be varied dependingon the intended use and/or to control the rate of lipase activity.

In certain embodiments, the device may be configured to allow a changein its size and or shape. For example, FIGS. 10A-10C illustrate a device1000, which may be compressed, e.g., for storage in a container 1001before use. When desired, the container 1001 can be opened, and the wall1003 of the device can be expanded to produce a desired ratio of lipasevolume 1002 to container volume. In some embodiments, the device 1000includes a coil or spring 1004 to provide structural support and/or tohelp the device maintain a desired shape and/or volume.

In some embodiments, the device can include a rod-like extension tofacilitate placement and removal of lipase within a volume of formula.For example, FIGS. 11A-11C, 12, 13A-13B, 14, and 15 illustrate variousexemplary configurations for devices with a rod-like extension. Asshown, the device 1100, 1100′, 1100″, 1200, 1300, 1400, 1500 can includeeither one or multiple pockets or openings 1101, 1201, 1301, 1401, 1501positioned in various configurations near a distal region of therod-like extension 1102, 1202, 1302, 1402, 1502. In some embodiments, asshown for example, in FIGS. 13A-13B, the orientation of the pockets oropenings 1301 can be adjustable, e.g., to allow insertion within anarrow opening during use and/or to minimize storage space.

In various embodiments, lipase may be attached to a portion of a cap orclosure for a bottle or jar, such that when the cap or closure is placedon the bottle or jar, the lipase may contact fluids contained within thebottle or jar. For example, any of the devices shown herein may beattached to a surface of a cap or closure to allow contact with formulacontainer in a bottle or jar. Various configurations of devices 1600,1700, 1800, 1900, 2000 including lipase attached to a cap or closure1602, 1702, 1802, 1902, 2002 are illustrated in FIGS. 16, 17A-17B, 18,19, and 20. As shown, the lipases can be contained within pockets oropenings 1601, 1705, 1805, 1901, 2001 having a variety of shapes orconfigurations. Further, in some embodiments, the cap or closure 1902,2002 can include an opening 1910, 2010 for inserting or removing fluidfrom a container, and such openings 1910, 2010 can include a connectorfor a fluid tube, e.g., a luer-type connector.

In some embodiments, it may be desirable to treat formula as the formulaflows through a tube (e.g., during feeding, as shown in FIG. 1, orduring transfer from one container to another). FIG. 21 illustratesanother device 2100 for in-line treatment of lipases. The device 2100can include a pocket or opening 2101 containing lipases, which may beimmobilized, as discussed above. Further, the pocket or opening 2101 mayhave a tortuous or curved path to allow for longer contact times betweenthe lipases and formula. In addition, the device 2100 may includeopenings 2110 at both ends to allow connection to tubes or conduits foringress and egress of formula.

In various embodiments, the devices may include a material that acts asa screen or mesh to prevent lipases from entering the formula to beingested by a patient. For example, the devices shown in FIGS. 19 and 21can include one or more meshes or screens 1906, 2106 to prevent lipasesimmobilized on beads or other structures from moving into formula to beingested.

In some embodiments, lipases can be immobilized within or on a componentof a container such that the lipases are not in contact with formulauntil further steps are taken. For example, in one embodiment, lipasesmay be contained within or on a portion of a cap or closure, and the capor closure may include a mechanism for releasing immobilized lipasesinto the container. For example, lipases may be contained on or withinbeads or other structures (see, e.g., element 1805 in FIG. 18), that arefurther attached to or contained within the cap; and when desired, thelipases may be dropped into the container (e.g., by twisting the cap orremoving a barrier/attachment mechanism). Similarly, lipases may beattached to or contained with a wall of the container or other structureand immobilized on beads or other materials, and the lipases can beallowed to contact formula only when desired (e.g., by releasing lipasesinto the container or removing a barrier over the lipases).

FIG. 6A is a photograph of a vial containing Rhizopus oryzae lipaseimmobilized on polymer beads. The immobilized lipase is in dry granularform which may be added to the container or chamber of a deviceaccording to the invention, such as the device depicted in FIGS. 6B-D.The immobilized lipase may be trapped in the chamber of the device,while still allowing the flow of formula through and out of the chamberby simply providing a filter at the egress end of the chamber thatcontains pores sufficiently large to allow formula to pass but retainsthe immobilized lipase within the chamber. Alternatively, lipase may beimmobilized by, e.g., coating the inner channels or chamber of thedevice so that the formula is exposed to the lipase as it passes throughthe chamber. The immobilized lipase in such a device may be used forcontinuous feeding for extended periods because of the increasedstability and reusability of the lipase.

Nutritional Formulas

Certain embodiments of the invention provide nutritional formulas. Insome embodiments, the nutritional formula is an infant formula. In someembodiments, the nutritional formula is a medical nutritional formula.In some embodiments, a nutritional formula is exposed to lipase prior toingestion. In some embodiments, this exposure allows pre-hydrolysis ofat least some lipids in the nutritional formula. Thus, in someembodiments, a nutritional formula is an “as-fed” formula, i.e., theliquid formula as composed just prior to ingestion by the subject, whichdiffers in composition from the formula as sold by the manufacturer. Theterm “nutritional formula” does not encompass compositions existingwithin the body of a subject after ingestion.

In some embodiments, the nutritional formula comprises long-chain fattyacids. In some embodiments, the nutritional formula comprises one ormore LC-PUFAs, such as DHA, ARA, and EPA. In some embodiments, thenutritional formula comprises DHA. In some embodiments, the nutritionalformula comprises ARA. In some embodiments, the nutritional formulacomprises DHA and ARA. In some embodiments, the nutritional formulacomprises DHA, ARA, and EPA.

In some embodiments, more than 5% of the total long-chain fatty acids inthe nutritional formula are in the form of monoglycerides and/or freefatty acids. In some embodiments, more than 5% of the total LC-PUFA inthe nutritional formula is in the form of monoglycerides and/or freefatty acids. In some embodiments, more than 5% of the DHA is in the formof a monoglyceride and/or a free fatty acid. In some embodiments, morethan 5% of the ARA is in the form of a monoglyceride and/or a free fattyacid. In some embodiments, more than 5% of the EPA is in the form of amonoglyceride and/or a free fatty acid.

In some embodiments, more than 10%, more than 15%, more than 20%, morethan 30%, more than 40%, more than 50%, more than 60%, more than 70%,more than 80%, more than 85%, more than 90%, more than 95%, or 100% ofthe total long-chain fatty acids in the nutritional formula are in theform of monoglycerides and/or free fatty acids. In some embodiments,more than 10%, more than 15%, more than 20%, more than 30%, more than40%, more than 50%, more than 60%, more than 70%, more than 80%, morethan 85%, more than 90%, more than 95%, or 100% of the total LC-PUFA inthe nutritional formula is in the form of monoglycerides and/or freefatty acids. In some embodiments, more than 10%, more than 15%, morethan 20%, more than 30%, more than 40%, more than 50%, more than 60%,more than 70%, more than 80%, more than 85%, more than 90%, more than95%, or 100% of the DHA is in the form of a monoglyceride and/or a freefatty acid. In some embodiments, more than 10%, more than 15%, more than20%, more than 30%, more than 40%, more than 50%, more than 60%, morethan 70%, more than 80%, more than 85%, more than 90%, more than 95%, or100% of the ARA is in the form of a monoglyceride and/or a free fattyacid. In some embodiments, more than 10%, more than 15%, more than 20%,more than 30%, more than 40%, more than 50%, more than 60%, more than70%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%of the EPA is in the form of a monoglyceride and/or a free fatty acid.In some embodiments, more than 10%, more than 15%, more than 20%, morethan 30%, more than 40%, more than 50%, more than 60%, more than 70%,more than 80%, more than 85%, more than 90%, more than 95%, or 100% ofboth the DHA and the ARA is in the form of a monoglyceride and/or a freefatty acid. In a particular embodiment, more than 90% of both the DHAand the ARA is in the form of a monoglyceride and/or a free fatty acid.In a particular embodiment, more than 95% of both the DHA and the ARA isin the form of a monoglyceride and/or a free fatty acid.

In some embodiments, at least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, or 100% of the totallong-chain fatty acids in the nutritional formula are in the form ofmonoglycerides and/or free fatty acids. In some embodiments, at least10%, at least 15%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 95%, or 100% of the total LC-PUFA in the nutritionalformula is in the form of monoglycerides and/or free fatty acids. Insome embodiments, at least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, or 100% of the DHA is inthe form of a monoglyceride and/or a free fatty acid. In someembodiments, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100% of the ARA is in the formof a monoglyceride and/or a free fatty acid. In some embodiments, atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100% of the EPA is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, at least10%, at least 15%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 95%, or 100% of both the DHA and the ARA is in the form ofa monoglyceride and/or a free fatty acid. In a particular embodiment, atleast 90% of both the DHA and the ARA is in the form of a monoglycerideand/or a free fatty acid. In a particular embodiment, at least 95% ofboth the DHA and the ARA is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments of the invention, the approximate serving size of anutritional formula of the invention is about 100-110 mL for prematureinfant formula, 90-150 mL (e.g., 148 mL) for term infant formula,230-500 mL (e.g., 235-250 mL) for enteral feeds, and 230-250 mL forchild formulas and adult formulas. In some embodiments, each servingcontains about 10-35 mg of ARA free fatty acids and monoglycerides (aswould be obtained from complete hydrolysis of TG-ARA in currentlyavailable preterm and term infant formulas) or about 40-50 mg of ARAfree fatty acids and monoglycerides (as would be obtained from completehydrolysis of TG-ARA in a currently available adult formula). In someembodiments, each serving contains about 7-20 mg of DHA free fatty acidsand monoglycerides (as would be obtained from complete hydrolysis ofTG-DHA in currently available preterm and term infant formulas) or about10-40 mg of DHA free fatty acids and monoglycerides (as would beobtained from complete hydrolysis of TG-DHA in currently available childand adult formulas). In some embodiments, an adult serving of 230-250 mLcontains about 1,100 mg of EPA free fatty acids and monoglycerides andabout 240 mg of DHA free fatty acids and monoglycerides (as would beobtained from complete hydrolysis of TG-EPA and TG-DHA in some currentlyavailable adult formula, such as ProSure®. In some embodiments of theinvention, however, the ability to pre-hydrolyze TG-LCPUFAs beforeingestion allows formula to be made with higher levels of LC-PUFAs thanin currently available formulas. Accordingly, in some embodiments theamount of free fatty acids and/or monoglycerides of ARA and/or DHAexceeds the amounts that could be obtained from complete hydrolysis ofTG-LCPUFAs in currently available formulas. In some embodiments, aserving of a nutritional formula of the invention contains 50-100 mg ofLC-PUFA free fatty acids and/or monoglycerides. In some embodiments, aserving of a nutritional formula of the invention contains 100-200 mg ofLC-PUFA free fatty acids and/or monoglycerides. In some embodiments, aserving of a nutritional formula of the invention contains 200-300 mg ofLC-PUFA free fatty acids and/or monoglycerides. In some embodiments, aserving of a nutritional formula of the invention contains 250-500 mg ofLC-PUFA free fatty acids and/or monoglycerides. In some embodiments, aserving of a nutritional formula of the invention contains 500-1000 mgof LC-PUFA free fatty acids and/or monoglycerides. In some embodiments,a serving of a nutritional formula of the invention contains 1-2 gramsof LC-PUFA free fatty acids and/or monoglycerides. In some embodiments,a serving of a nutritional formula of the invention contains 2-3 gramsof LC-PUFA free fatty acids and/or monoglycerides.

In some embodiments, the nutritional formula comprises fats,carbohydrates, and proteins (or amino acids). In some embodiments, aninfant formula of the invention comprises one, more than one, or all ofthe following: nonfat milk, lactose, vegetable oil (e.g., one or more ofpalm olein, coconut, soy, and high oleic sunflower oils), whey proteinconcentrate, sugars, LC-PUFAs, vitamins, and minerals. In someembodiments, the nutritional formula comprises fats composed ofmedium-chain fatty acids and fats composed of long-chain fatty acids. Insome embodiments, the nutritional formula comprises fats composed of n-6fatty acids and fats composed of n-3 fatty acids. In some embodiments,the nutritional formula comprises LA and ALA.

In some embodiments, a nutritional formula of the invention does notcomprise added lipase. In some embodiments, a device and/or a method ofthe present invention is used to expose a nutritional formula to alipase, but the nutritional formula is separated from the lipase beforefeeding, such that the as-fed nutritional formula does not compriseadded lipase. A nutritional formula that does not comprise added lipaserefers to a formula in which lipase is not detectable or is present onlyat very low levels, due, e.g., to leaching of immobilized lipase from asolid support into the formula. In some embodiments, a nutritionalformula comprises no more than 0.02% (w/w) lipase, no more than 0.01%(w/w) lipase, no more than 0.005% (w/w) lipase, no more than 0.002%(w/w) lipase, no more than 0.001% (w/w) lipase, no more than 0.0005%(w/w) lipase, no more than 0.0002% (w/w) lipase, or no more than 0.0001%(w/w) lipase. In some embodiments, a nutritional formula comprises lessthan 0.02% (w/w) lipase, less than 0.01% (w/w) lipase, less than 0.005%(w/w) lipase, less than 0.002% (w/w) lipase, less than 0.001% (w/w)lipase, less than 0.0005% (w/w) lipase, less than 0.0002% (w/w) lipase,or less than 0.0001% (w/w) lipase.

In some embodiments, the nutritional formula comprises a lipase. In someembodiments, the lipase is selected from Chromobacterium viscosumlipase, Pseudomonas fluorescens lipase, Burcholderia cepacia lipase, andRhizopus oryzae lipase. In some embodiments, the lipase is selected fromChromobacterium viscosum lipase, Pseudomonas fluorescens lipase, andRhizopus oryzae lipase. In some embodiments, the lipase isChromobacterium viscosum lipase. In some embodiments, the lipase isPseudomonas fluorescens lipase. In some embodiments, the lipase isRhizopus oryzae lipase.

In some embodiments, a serving of the nutritional formula contains lessthan 5,000 units of lipase (with units assessed in a standard oliveassay, such as described in Pharmaceutical Enzymes: Properties and AssayMethods, R. Ruyssen and A. Lauwers (Eds) Scientific Publishing Company,Ghent, Belgium (1978)). In other embodiments, a serving of thenutritional formula contains less than 3,000 units of lipase. In someembodiments, a serving of the nutritional formula contains less than1,000 units. In certain embodiments, the formula containing less than5,000, less than 3,000, or less than 1,000 units of lipase per servingis an infant formula or a medical nutritional formula.

In some embodiments, the nutritional formula contains 0.01 mg to 1 gramof lipase per gram of total fat (whether in free fatty acid,monoglyceride, ester, or triglyceride form) in the nutritional formula.In some embodiments, the nutritional formula contains 0.1 to 500 mg oflipase per gram of total fat in the nutritional formula. In someembodiments, the nutritional formula contains 0.1 to 250 mg of lipaseper gram of total fat in the nutritional formula. In some embodiments,the nutritional formula contains 0.1 to 200 mg of lipase per gram oftotal fat in the nutritional formula. In some embodiments, thenutritional formula contains 0.1 to 150 mg of lipase per gram of totalfat in the nutritional formula. In some embodiments, the nutritionalformula contains 0.1 to 100 mg of lipase per gram of total fat in thenutritional formula. In some embodiments, the nutritional formulacontains 0.1 to 50 mg of lipase per gram of total fat in the nutritionalformula. In some embodiments, the nutritional formula contains 1 to 50mg of lipase per gram of total fat in the nutritional formula. In someembodiments, the nutritional formula contains 25 to 75 mg of lipase pergram of total fat in the nutritional formula. In some embodiments, thenutritional formula contains 1 to 100 mg of lipase per gram of total fatin the nutritional formula. In some embodiments, the nutritional formulacontains no more than 50 mg of lipase per gram of total fat in thenutritional formula.

In some embodiments, the nutritional formula contains 0.001 to 10 mg oflipase per milligram of total LC-PUFA (whether in free fatty acid,monoglyceride, ester, or triglyceride form) in the nutritional formula.In some embodiments, the nutritional formula contains 0.001 to 5 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.001 to 3 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.001 to 1 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.001 to 0.5 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.001 to 0.1 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.001 to 0.05 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.01 to 0.1 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.02 to 0.08 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains 0.04 to 0.06 mg oflipase per milligram of total LC-PUFA in the nutritional formula. Insome embodiments, the nutritional formula contains no more than 0.1 mgof lipase per milligram of total LC-PUFA in the nutritional formula.

In some embodiments, the nutritional formula contains 0.001 to 10 mg oflipase per milligram of total DHA (whether in free fatty acid,monoglyceride, ester, or triglyceride form) in the nutritional formula.In some embodiments, the nutritional formula contains 0.001 to 5 mg oflipase per milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 3 mg of lipaseper milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 1 mg of lipaseper milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.5 mg of lipaseper milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.1 mg of lipaseper milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.05 mg of lipaseper milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.01 to 0.1 mg of lipaseper milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.02 to 0.08 mg of lipaseper milligram of total DHA in the nutritional formula. In someembodiments, the nutritional formula contains 0.04 to 0.06 mg of lipaseper milligram of total DHA in the nutritional formula.

In some embodiments, the nutritional formula contains 0.001 to 10 mg oflipase per milligram of total ARA (whether in free fatty acid,monoglyceride, ester, or triglyceride form) in the nutritional formula.In some embodiments, the nutritional formula contains 0.001 to 5 mg oflipase per milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 3 mg of lipaseper milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 1 mg of lipaseper milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.5 mg of lipaseper milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.1 mg of lipaseper milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.05 mg of lipaseper milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.01 to 0.1 mg of lipaseper milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.02 to 0.08 mg of lipaseper milligram of total ARA in the nutritional formula. In someembodiments, the nutritional formula contains 0.04 to 0.06 mg of lipaseper milligram of total ARA in the nutritional formula.

In some embodiments, the nutritional formula contains 0.001 to 10 mg oflipase per milligram of total EPA (whether in free fatty acid,monoglyceride, ester, or triglyceride form) in the nutritional formula.In some embodiments, the nutritional formula contains 0.001 to 5 mg oflipase per milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 3 mg of lipaseper milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 1 mg of lipaseper milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.5 mg of lipaseper milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.1 mg of lipaseper milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.001 to 0.05 mg of lipaseper milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.01 to 0.1 mg of lipaseper milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.02 to 0.08 mg of lipaseper milligram of total EPA in the nutritional formula. In someembodiments, the nutritional formula contains 0.04 to 0.06 mg of lipaseper milligram of total EPA in the nutritional formula.

In some embodiments, a nutritional formula is prepared by a methoddisclosed herein. In some embodiments, a nutritional formula is preparedusing a device disclosed herein.

Methods of Preparing a Nutritional Formula

According to various embodiments, the present disclosure also providesmethods of preparing nutritional formulas. In some embodiments, thenutritional formula is an infant formula. In some embodiments, thenutritional formula is a medical nutritional formula. In someembodiments, the nutritional formula is a nutritional drink for adults(such as a complete nutritional drink, e.g., ENSURE, PEDIASURE).

In some embodiments, a method of preparing a nutritional formulacomprises exposing a liquid nutritional composition to a lipase. In someembodiments, the liquid nutritional composition comprises LC-PUFAtriglycerides or LC-PUFA esters. In some embodiments, the liquidnutritional composition comprises triglycerides or esters of one or moreLC-PUFAs selected from the group consisting of DHA, ARA, and EPA.

In some embodiments, the liquid nutritional composition is exposed to alipase selected from Chromobacterium viscosum lipase, Pseudomonasfluorescens lipase, Burcholderia cepacia lipase, and Rhizopus oryzaelipase. In some embodiments, the lipase is selected from Chromobacteriumviscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzaelipase. In some embodiments, the lipase is Chromobacterium viscosumlipase. In some embodiments, the lipase is Pseudomonas fluorescenslipase. In some embodiments, the lipase is Rhizopus oryzae lipase.

Components involved in these methods may be mixed in various orders. Insome embodiments, lipase is added to a liquid nutritional composition,thereby exposing lipids in the liquid nutritional composition to thelipase. In some embodiments, a liquid nutritional composition isprepared by adding a potable liquid to a solid or powder form of thenutritional composition. In some embodiments, lipase is present in thesolid or powder form of the nutritional composition before the additionof potable liquid. In other embodiments, lipase is added after theliquid nutritional composition is prepared. In some embodiments, thelipase and the solid or powder form of the nutritional composition areadded to a potable liquid at the same time.

In some embodiments, the liquid nutritional composition is exposed tolipase for at least one minute, at least 2 minutes, at least 3 minutes,at least 5 minutes, at least 8 minutes, at least 10 minutes, at least 15minutes, at least 30 minutes, at least 45 minutes, or at least 60minutes prior to ingestion. In some embodiments, the liquid nutritionalcomposition is exposed to lipase for no more than 30 seconds, no morethan 1 minute, no more than 2 minutes, no more than 3 minutes, no morethan 5 minutes, no more than 8 minutes, no more than 10 minutes, no morethan 15 minutes, no more than 30 minutes, no more than 45 minutes, nomore than 60 minutes, no more than 2 hours, no more than 4 hours, nomore than 6 hours, no more than 12 hours, or no more than 24 hours.

In some embodiments, the method results in a nutritional formula inwhich at least 10%, at least 15%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, or 100% of the total LC-PUFA in thenutritional formula is in the form of monoglycerides and/or free fattyacids. In some embodiments, at least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% of the DHAis in the form of a monoglyceride and/or a free fatty acid. In someembodiments, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100% of the ARA is in the formof a monoglyceride and/or a free fatty acid. In some embodiments, atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100% of the EPA is in the form of amonoglyceride and/or a free fatty acid.

For purposes of this application, exposure of a nutritional compositionor formula to a lipase refers to the period of time in which a liquidnutritional composition or liquid formula is in contact with a lipase,which may be in solution or immobilized. For purposes of thisapplication, exposure to a lipase ends when the formula is ingested by asubject or when the lipase is removed by separating the liquid formulafrom a solid support to which the lipase is immobilized. In someembodiments, the liquid nutritional composition is exposed to lipase forno more than 10 minutes, and at least 20% of the DHA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid. In some embodiments, the liquid nutritional composition isexposed to lipase for no more than 10 minutes, and at least 20% of theARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 10minutes, and at least 20% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 10 minutes, and at least 40% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 10 minutes, and at least 40% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 10minutes, and at least 40% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 10 minutes, and at least 50% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 10 minutes, and at least 50% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 10minutes, and at least 50% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 10 minutes, and at least 60% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 10 minutes, and at least 60% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 10minutes, and at least 60% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 10 minutes, and at least 70% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 10 minutes, and at least 70% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 10minutes, and at least 70% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 20 minutes, and at least 20% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 20 minutes, and at least 20% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 20minutes, and at least 20% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 20 minutes, and at least 40% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 20 minutes, and at least 40% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 20minutes, and at least 40% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 20 minutes, and at least 50% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 20 minutes, and at least 50% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 20minutes, and at least 50% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 20 minutes, and at least 80% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 20 minutes, and at least 80% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 20minutes, and at least 80% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 20 minutes, and at least 90% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 20 minutes, and at least 90% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 20minutes, and at least 90% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 30 minutes, and at least 20% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 30 minutes, and at least 20% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 30minutes, and at least 20% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 30 minutes, and at least 40% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 30 minutes, and at least 40% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 30minutes, and at least 40% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 30 minutes, and at least 60% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 30 minutes, and at least 60% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 30minutes, and at least 60% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 30 minutes, and at least 70% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 30 minutes, and at least 70% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 30minutes, and at least 70% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the liquid nutritional composition is exposed tolipase for no more than 30 minutes, and at least 80% of the DHA in theresulting nutritional formula is in the form of a monoglyceride and/or afree fatty acid. In some embodiments, the liquid nutritional compositionis exposed to lipase for no more than 30 minutes, and at least 80% ofthe ARA in the resulting nutritional formula is in the form of amonoglyceride and/or a free fatty acid. In some embodiments, the liquidnutritional composition is exposed to lipase for no more than 30minutes, and at least 80% of the total LC-PUFA in the resultingnutritional formula is in the form of a monoglyceride and/or a freefatty acid.

In some embodiments, the lipase remains in the nutritional formula whenit is fed to the subject. In other embodiments, the lipase is removedfrom the liquid nutritional composition before it is fed to the subject.In some embodiments, the lipase is removed by exposing the liquidnutritional composition comprising the lipase to a solid supportimmobilized to a molecule that binds to the lipase, thereby binding thelipase to the solid support, and separating the liquid nutritionalcomposition from the solid support. Since the lipase is immobilized tothe solid support, separating the liquid nutritional composition fromthe solid support has the effect of removing the lipase from the liquidnutritional composition. In some embodiments, the lipase is immobilizedto a solid support before it is exposed to the liquid nutritionalcomposition, and the lipase is removed by separating the liquidnutritional composition from the solid support. In some embodiments, thelipase is immobilized to at least a portion of an interior face of achamber or to a solid support contained within the chamber, and theliquid nutritional composition is temporarily exposed to the lipase bypassing through the chamber. In some embodiments, the chamber is acolumn. In some embodiments, the liquid nutritional composition isexposed to a container containing lipase immobilized to a solid support,and at least a portion of the inner surface of the container consists ofa material that is permeable to triglycerides and esters but is notpermeable to the solid support.

In some embodiments, the method produces a nutritional formula that doesnot comprise added lipase. In some embodiments, a nutritional formula isexposed to a lipase, but the nutritional formula is separated from thelipase before feeding, such that the as-fed nutritional formula does notcomprise added lipase. A nutritional formula that does not comprise (orcontain) added lipase refers to a formula in which lipase is notdetectable or is present only at very low levels, due, e.g., to leachingof immobilized lipase from a solid support into the formula. In someembodiments, a nutritional formula comprises no more than 0.02% (w/w)lipase, no more than 0.01% (w/w) lipase, no more than 0.005% (w/w)lipase, no more than 0.002% (w/w) lipase, no more than 0.001% (w/w)lipase, no more than 0.0005% (w/w) lipase, no more than 0.0002% (w/w)lipase, or no more than 0.0001% (w/w) lipase. In some embodiments, anutritional formula comprises less than 0.02% (w/w) lipase, less than0.01% (w/w) lipase, less than 0.005% (w/w) lipase, less than 0.002%(w/w) lipase, less than 0.001% (w/w) lipase, less than 0.0005% (w/w)lipase, less than 0.0002% (w/w) lipase, or less than 0.0001% (w/w)lipase.

In some embodiments, the method comprises exposing the nutritionalformula to less than 5,000 units of lipase per serving (with unitsassessed in a standard olive assay, such as described in PharmaceuticalEnzymes: Properties and Assay Methods, R. Ruyssen and A. Lauwers (Eds)Scientific Publising Company, Ghent, Gelgium (1978)). In otherembodiments, the nutritional formula is exposed to less than 3,000 unitsof lipase per serving. In some embodiments, the nutritional formula isexposed to less than 1,000 units of lipase per serving. In certainembodiments, the formula exposed to less than 5,000, less than 3,000, orless than 1,000 units of lipase per serving is an infant formula or amedical nutritional formula.

In some embodiments, a method of the invention exposes the nutritionalformula to 0.01 mg to 1 gram of lipase per gram of total fat (whether infree fatty acid, monoglyceride, ester, or triglyceride form) in thenutritional formula. In some embodiments, a method of the inventionexposes the nutritional formula to 0.1 to 500 mg of lipase per gram oftotal fat in the nutritional formula. In some embodiments, a method ofthe invention exposes the nutritional formula to 0.1 to 250 mg of lipaseper gram of total fat in the nutritional formula. In some embodiments, amethod of the invention exposes the nutritional formula to 0.1 to 200 mgof lipase per gram of total fat in the nutritional formula. In someembodiments, a method of the invention exposes the nutritional formulato 0.1 to 150 mg of lipase per gram of total fat in the nutritionalformula. In some embodiments, a method of the invention exposes thenutritional formula to 0.1 to 100 mg of lipase per gram of total fat inthe nutritional formula. In some embodiments, a method of the inventionexposes the nutritional formula to 0.1 to 50 mg of lipase per gram oftotal fat in the nutritional formula. In some embodiments, a method ofthe invention exposes the nutritional formula to 1 to 50 mg of lipaseper gram of total fat in the nutritional formula. In some embodiments, amethod of the invention exposes the nutritional formula to 25 to 75 mgof lipase per gram of total fat in the nutritional formula. In someembodiments, a method of the invention exposes the nutritional formulato 1 to 100 mg of lipase per gram of total fat in the nutritionalformula. In some embodiments, a method of the invention exposes thenutritional formula to no more than 50 mg of lipase per gram of totalfat in the nutritional formula.

In some embodiments, the method exposes the nutritional formula to 0.001to 10 mg of lipase per milligram of total LC-PUFA (whether in free fattyacid, monoglyceride, ester, or triglyceride form) in the nutritionalformula. In some embodiments, the nutritional formula is exposed to0.001 to 5 mg of lipase per milligram of total LC-PUFA in thenutritional formula. In some embodiments, the nutritional formula isexposed to 0.001 to 3 mg of lipase per milligram of total LC-PUFA in thenutritional formula. In some embodiments, the nutritional formula isexposed to 0.001 to 1 mg of lipase per milligram of total LC-PUFA in thenutritional formula. In some embodiments, the nutritional formula isexposed to 0.001 to 0.5 mg of lipase per milligram of total LC-PUFA inthe nutritional formula. In some embodiments, the nutritional formula isexposed to 0.001 to 0.1 mg of lipase per milligram of total LC-PUFA inthe nutritional formula. In some embodiments, the nutritional formula isexposed to 0.001 to 0.05 mg of lipase per milligram of total LC-PUFA inthe nutritional formula. In some embodiments, the nutritional formula isexposed to 0.01 to 0.1 mg of lipase per milligram of total LC-PUFA inthe nutritional formula. In some embodiments, the nutritional formula isexposed to 0.02 to 0.08 mg of lipase per milligram of total LC-PUFA inthe nutritional formula. In some embodiments, the nutritional formula isexposed to 0.04 to 0.06 mg of lipase per milligram of total LC-PUFA inthe nutritional formula. In some embodiments, the nutritional formula isexposed to no more than 0.1 mg of lipase per milligram of total LC-PUFAin the nutritional formula.

In some embodiments, the method exposes the nutritional formula to 0.001to 10 mg of lipase per milligram of total DHA (whether in free fattyacid, monoglyceride, ester, or triglyceride form) in the nutritionalformula. In some embodiments, the nutritional formula is exposed to0.001 to 5 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to0.001 to 3 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to0.001 to 1 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to0.001 to 0.5 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to0.001 to 0.1 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to0.001 to 0.05 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to 0.01to 0.1 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to 0.02to 0.08 mg of lipase per milligram of total DHA in the nutritionalformula. In some embodiments, the nutritional formula is exposed to 0.04to 0.06 mg of lipase per milligram of total DHA in the nutritionalformula.

In some embodiments, a method of preparing a nutritional formulacomprises exposing a liquid nutritional composition to a device asdescribed herein.

Example 1: Specific Activities of Lipases for DHA and ARA

To evaluate the enzymatic activity of various lipases on DHA and/or ARAtriglycerides, experiments were performed in a two mL glass vial (withmagnetic stir bar) containing 0.1M Tris buffer, pH 7.7 and the substrateDHA or ARA triglycerides. The reaction was initiated by adding lipasesolutions. Lipases were obtained from commercial sources as follows:Rhizopus oryzae (Amano DF-15, Amano Enzymes Inc., Nagoya, Japan),Chromobacterium viscosum (EMD CalBiochem, EMD Biosciences, Billerica,Mass.), and Pseudomonas fluoresens (Amano AK, Amano Enzymes Inc.,Nagoya, Japan). Other lipases are also available from commercialsources, such as Candida rugosa (Amano AY 30 or Amano 30, Amano EnzymesInc., Nagoya, Japan), Aspergillus niger (Amano DS, Amano Enzymes Inc.,Nagoya, Japan), Penicillium camembertii (Amano 50, Amano Enzymes Inc.,Nagoya, Japan), Rhizomucor miehei (L4277, Sigma-Aldrich), Asperigillusoryzae (62285, Sigma-Aldrich), and Burcholderia cepacia (534641,Sigma-Aldrich). Lipase solutions were prepared from these commerciallyavailable lipases without additional purification, except that B.cepacia lipase was purified to homogeneity.

The vials were transferred to a water-bath at 37° C. placed on amagnetic stirrer. 50 μl of samples were taken at different timeintervals—0, 15, 30, 45, 60, 90, and 120 min and added to a HPLC vialcontaining 950 μl of running buffer (30% 10 mM ammonium phosphatebuffer, pH 3.0 and 70% acetonitrile). The samples were then analyzed foreither DHA free acid or ARA free acid by reverse phase high performanceliquid chromatography (RP-HPLC) using an Agilent HPLC1100 series and aC8 RP column and monitoring at 215 and 220 nm. The free acid peaks wereidentified according to retention times using commercially availablestandards: DHA triglyceride (Nu-check Prep, Inc. Lot No. T-310-D7-V),ARA triglyceride (Nu-check Prep, Inc. Lot No. T-295-JY14-V), DHA freeacid form (Nu-check Prep, Inc. Lot No. U-84A-AU20-U), and ARA free acidform (Nu-check Prep, Inc. Lot No. U-71A-N11-U). The specific activitiesof a panel of lipases in this assay for DHA and ARA are summarized inTable 1. In the inventors' hands, Chromobacterium viscosum (CV),Burcholderia cepacia (BC), Pseudomonas fluoresens (PF), and Rhizopusoryzae (RO) had substantially higher specific activity toward DHA and/orARA compared to the other lipases tested, including Candida rugosa (CR).

TABLE 1 Specific activities of lipases for DHA and ARA DHA produced, ARAproduced, Enzyme Πmol/min/mg Πmol/min/mg CV 27.73 23.207 PF 4.66 2.599RO 9.83 6.297 CR 0.01 0.205 AN 0.00 0.000 PC 0.00 0.000 BC 55.11 13.920AO 0.33 0.263 RM 0.14 0.03

Example 2: Enzymatic Activity of Chromobacterium viscosum and Rhizopusoryzae Lipases for DHA, ARA, and EPA in Infant Formula

To evaluate the enzymatic activity of CV and RO lipase on DHA, ARA, andEPA when supplemented to infant formula, milk-based infant formula wasprepared by dissolving 10 g of ENFAMIL® powder in 35 mL of water. Infantformula containing substrate EPA, 0.1 M Tris buffer, pH 7.7, andsubstrates 2.7 mg DHA (Nu-check Prep, Inc. Lot No. T-310-D7-V) and 5.4mg ARA (Nu-check Prep, Inc. Lot No. T-295) were added to a one mL glassvial (with magnetic stir bar). The reaction was initiated by addingenzyme (i.e. lipase); four concentrations of each enzyme were tested.The vials were transferred to a water bath at 37° C. placed on amagnetic stirrer. 50 μl of each sample were taken at different timepoints—0, 10, 20, 30, 45, and 60 min and added to a HPLC vial containing950 μl of HPLC running buffer (30% 10 mM ammonium phosphate buffer, pH3.0 and 70% acetonitrile). The samples were then analyzed for either DHAacid, ARA acid, or EPA acid by RP-HPLC as above.

The percent of total triglycerides decreased over time as the amount offree acid and monoglyceride increased. For example, when hydrolyzed withRO, the amount of DHA free acid increased with time (FIG. 7). Similarly,when hydrolyzed with RO, the amount of ARA free acid increased with time(FIG. 8).

The specific activities of each of the lipases in this assay werecalculated based on the amount of free DHA acid, ARA acid, or EPA acidreleased in the infant formula and are shown in Table 2.

TABLE 2 Specific activities of lipases on TG-DHA, TG-ARA, and TG-EPA ininfant formula DHA produced, ARA produced, EPA produced, Enzymeμmol/min/mg μmol/min/mg μmol/min/mg CV 39.461 36.587 19.866 RO 13.06210.940 14.156

Example 3: Hydrolysis of DHA Triglyceride and ARA Triglyceride Scale Up

Lipases were evaluated for their ability to hydrolyze TG-DHA and TG-ARAwhen scaled up to an amount that may be used for supplementing infantformula. The infant formula (milk) was prepared by dissolving 162 g ofEnfamil powder in 648 mL of tap water (hot water, the temperature was37° C.). DHA triglyceride (442 mg, final concentration of DHA was 0.54g, 1.2% of total fat) and ARA triglyceride (885 mg, final concentrationARA 1.08 g, 2.4% of total fat) were accurately weighed from the samesource as in Example 2, and were mixed with the infant formula powderbefore adding the water. The reaction was carried out in a water-bathwith constant stirring. Fat hydrolysis was initiated by adding either CVor RO lipase. Formula samples were withdrawn at 0, 15, and 30 minutesand were analyzed for hydrolysis of DHA and ARA by RP-HPLC, as describedabove. The results are shown in Table 3 below.

TABLE 3 Hydrolysis of TG-DHA and TG-ARA in infant formula % Hydrolysis %Hydrolysis Lipase, DHA ARA mg 15 min 30 min 15 min 30 min CV 9 75.1771.32 58.97 74.38 18 84.51 87.25 65.83 63.31 RO 29 62.35 71.19 104.81124.67 58 73 67.77 124.22 113.08

Example 4: Enzymatic Activity of Immobilized Rhizopus oryzae Lipase onTG-DHA or TG-ARA in Infant Formula and Buffer

To evaluate the enzymatic activity of immobilized RO lipase on TG-DHA orTG-ARA when supplemented to infant formula, milk-based infant formulawas prepared by dissolving 10 g of ENFAMIL® powder in 35 mL of water.The reaction was carried out as follows. Infant formula, 0.1M Trisbuffer, pH 7.7, and substrate (TG-DHA or TG-ARA) were added to a one mLglass vial (with magnetic stir bar). The reaction was initiated byadding lipase. The vials were transferred to a water bath at 37° C.placed on a magnetic stirrer. 50 μl of each sample were taken atdifferent time points—0, 10, 20, and 30 min and added to a HPLC vialcontaining 900 μl of HPLC running buffer (30% 10 mM ammonium phosphatebuffer, pH 3.0 and 70% acetonitrile). The samples were then analyzed foreither DHA acid or ARA acid by RP-HPLC as described above.

The specific activities of the lipase for hydrolysis of TG-DHA andTG-ARA were calculated based on the amount of free DHA acid or ARA acidreleased in the infant formula and are shown in Table 4.

TABLE 4 Specific activities of Immobilized RO lipase on TG-DHA or TG-ARAin infant formula and Buffer DHA produced, ARA produced, Enzymeμmol/min/mg μmol/min/mg Immobilized Rhizopus 0.017 (0.004) 0.020 (0.008)oryzae lipase (RO) The values in parentheses are for buffer alone.

Example 5: Animals and Surgical Procedure 5.1 Animals

The experiments were performed on 12 pigs (9+3) from the University herdat Odarslöv, Swedish Agricultural University, Department of AgriculturalBiosystems and Technology, weighing approximately 10±2 kg each. Animalswere maintained on a 12 hour day-night cycle, with light from06.00-18.00 (6 am-6 pm) and dark from 18.00-06.00 (6 pm-6 am) hours. Thepigs were individually housed in metabolic cages or individual pensequipped with a dry feeding trough, a drinking nipple and a constantheating lamp (150 W). They were allowed to move freely within their pen,and have visual contact with each other.

5.2 Feed

Following surgery and during the pre-treatment period, pigs were fed astandard pig diet (“53908 Växtill 320 P BK”, Lantmännen, Sweden)containing 17.5% crude protein, 3.9% crude fibre, 3.5% crude fat, and5.2% ash together with 5000 IE/kg vitamine A, 500 IE/kg vitamine D, and85 mg/kg vitamine E. Pigs were fed twice daily (2.0% of body mass permeal) at 09:00-10:00 hr (9 am-10 am) and 17:00-18:00 hr (5 pm-6 pm). Fora few days before the start of the experiment, i.e., before theadaptation period, pigs were trained to consume infant formula (NAN Pro1 Gold Infant Formula, Nestle). Formula was prepared as a 1:4 dilutionin tap water instead of 1:7 as recommended by manufacturer to allowproper consumption, since pigs do not like to drink large volumes ofliquid. Daily nutrient requirements are 400 kJ/kg body weight,corresponding 40 g of formula powder/kg body weight. Daily feed wasdivided into 4 portions, starting with the first meal at 9 am and than,every 3 h after with the last meal of day at 6 pm. 100 g of NAN formulacontains about 27.7% fat, 9.6% protein, and 57.8% carbohydrates.

5.3 Infant Formula Milk Fortified with DHA and ARA Triglycerides

According to the manufacturer, NAN Pro 1 Gold (Nestle) is a premium wheypredominant starter infant formula that is nutritionally complete andspecially formulated for healthy infants from birth. It also containsfish oil to help support brain and visual development.

(http://www.nestlebaby.com/au/baby_nutrition/products/infant_formula/)

NAN Pro 1 Gold Ingredients:

-   -   Milk solids, vegetable oils (contains soy), minerals (calcium        citrate, potassium citrate, potassium chloride, magnesium        chloride, sodium chloride, sodium sulphate, ferrous sulphate,        zinc sulphate, calcium phosphate, copper sulphate, manganese        sulphate, potassium iodide, sodium selenate), omega LCPUFAs (DHA        from fish oil, AA), emulsifier (soy lecithin), vitamins [sodium        ascorbate (vit C), d-l alpha tocopheryl acetate (vit E),        niacinamide (niacin), calcium pantothenate, retinyl acetate (vit        A), thiamine mononitrate (vit B1), pyridoxine hydrochloride (vit        B6), riboflavin (vit B2), folic acid, phylloquinone (vit K1),        biotin, cholecalciferol (vit D3)], cyanocobalamin (B 12),        L-histidine, taurine, inositol, nucleotides (cytidine        5′-monophosphate, uridine 5′-monophosphate, adenosine        5′-monophosphate, guanosine 5′-monophosphate), L-carnitine,        culture (bifidus).        Table 5 below summarizes the lipid composition of human milk and        infant formula milk*, together with infant formula and pig        formula milk for use in these experiments.

TABLE 5 LCPUFA fortified formula milk Infant pre- Pig formula HumanInfant term term for study in milk* formula formula EPI pigs TG g/L25-29 33-36 25 50 Cholesterol  9-15 0-4 10 mg/dL In % of total FAPalmitate 20-25  8-24 NA* NA* C16:0 Sterate  7-12 2-6 NA* NA* C18:0 LA 10-15.5  10-29.9 4.5 NA* C18:2n-6 LNA 0.58-1.44 0.08-2   0.086 NA*C18:3n-3 DHA 0.19-0.42   0-0.15 0.083 Up to C22:6n-3 1.6%** (ω 3) ARA 0.4-0.54  0-0.4 0.167* Up to C20:4n-6 3.15%** (ω 6) *Data from variouswomen cohorts in Australia, Europe, United States, and Canada between1990 and 2005. Data from various infant term formulas including Nutrilom(Nutricia, Netherland), Enfamil (Mead Johnson, Canada), Similac (AbbottRoss, US), SMA (Wyeth, US). (JPGN 2010; 51: 380-401)// **Huang M C,2007, ^($$)MCT 9.6 *Final concentration will be measured afterexperiment is finishedThe total concentration of TG-DHA and TG-AA in NAN formula is 0.22%,which is below the recommended levels of 1%. Thus, NAN formula wasfortified with TG-DHA and TG-AA from fish oil (NuCheck(http://www.nu-chekprep.com, ˜40% TG-DHA and TG-ARA) to reach a finalconcentration of 1% TG-DHA and 2% TG-DHA respectively.

5.4 Pancreatic Duct Ligation Surgery for Induction of ExocrinePancreatic Insufficiency (EPI)

EPI surgery was performed on 12+2 young pigs 6-8 weeks of age. EPI istypically fully developed three to four weeks after the surgery.Development of total pancreatic insufficiency was confirmed by arrestedgrowth (minimal or no increase in body weight) and/or development ofsteathorrhea.

Example 6: Experimental Design, Procedures 6.1 Study Design

The study contained three periods: adaptation, control, and testing.During the 7-day adaptation period, pigs were trained to drink infantformula fortified with TG-DHA and TG-ARA. During the 7-day controlperiod, pigs continued to be fed infant formula fortified with TG-DHAand TG-ARA. During the 7-day testing period, the pigs were fed infantformula fortified with TG-DHA and TG-ARA, either (a) non-hydrolyzed; (b)pre-hydrolyzed with CV lipase; or (c) pre-hydrolyzed with RO lipase.Formula consumption was measured daily, faeces samples were collectedthe last 3 days of each study period (72 hr collection), and bloodsamples were collected on day 7 of the control and testing periods.

6.2 Lipase Dosing

Dose of lipase and pre-hydrolysis time were determined based on in vitroresults (Example 3) and daily nutritional requirements of pigs. NANformula mixed with lipase RO or CV (˜1300 U/g total fat) was incubatedwith shaking for 15 minutes at 37 C.

Proposed Formula Preparation and Lipase Mix:

-   -   Body weight of pigs ranged from 11-14 kg    -   Feed requirement: 40 g formula powder/kg body weight;    -   Daily need 500 g powder/pig    -   4 meals per day    -   125 g powder/pig/meal

Meal Preparation: 500 d Powder+1.5 L Water (Dilution 1:4)

-   -   Add dry powder first    -   Add TG-PUFA oils (7.5 mL DHA and 15 mL ARA), mix well, and add        tap water from water bath at 37 C, mix well    -   For lipase-treated formula, mix in CV or RO lipase    -   Add water to final volume    -   Mix all 15 min in water bath at 37 C    -   Divide into 4 buckets and feed each pig ˜600 mL of formula

6.2.1 Adaptation Period (7-10 Days)

Approximately 7-10 days before the Adaptation period, 12 pigs wereplaced in metabolic cages and trained to drink formula enriched withTG-PUFA. On the first morning of the Adaptation period, body weight wasrecorded before the morning meal.

6.2.2 Control Period (7 Days)

To all selected pigs infant formula was given as the only source offood, 4 times per day. The total daily formula consumption was measuredduring the entire experiment. On the morning of the first day of theControl period, body weight was recorded before the morning meal. 3×24hr stool samples were collected from day 5 through day 7. Blood sampleswere collected on the last day of this period, 1 hr before a meal and 1,2, and 3 hours later.

6.2.3 Testing Period (7 Days)

To all selected pigs TG-PUFA enriched infant formula was given as theonly source of food, 4 times per day. The total daily milk consumptionwas measured during the entire experiment. On the morning of the firstday of the Testing period body weight was recorded before morning meal.3×24 h stool samples were collected from day 5 through day 7. Bloodsamples were collected on the last day of this period, 1 h before a mealand 1, 2, and 3 h later.

Before the start of this period, pigs were randomized into three groups,based on body weight and willingness to drink formula:

1) One-third of the EPI pigs (n=4) were fed formula pre-hydrolyzed withRO lipase;

2) One-third of the EPI pigs (n=4) were fed formula pre-hydrolyzed withCV lipase; and

3) One-third of the EPI pigs (n=4) were fed formula only.

Preparation of the formula and lipase mix is shown above.

6.3 Criteria for a Positive Response

Significant reduction in fecal LCPUFA, increase in coefficient of fatabsorption (% CFA), and increase in concentration of plasma LCPUFA whencompared to EPI pigs fed only formula supplemented with 2% TG-ARA and 1%TG-AA.

6.4 Data Analysis

Individual data were recorded at the time they are generated.Statistical analyses were performed using the Student t-test.Differences were considered significant if p<0.05.

Example 7: RO and CV Lipases Improve Fatty Acid Absorption in EPI Pigs

Pigs with exocrine pancreatic insufficiency (EPI), a well establishedsurgical model, were used as a model to mimic pre-term or full-termhuman babies, where exocrine pancreatic function is compromised. The EPIsurgical pig model was used essentially as described in Examples 5 and 6to evaluate the effect on fatty acid absorption of infant formulapre-hydrolyzed with CV lipase or RO lipase as compared to non-hydrolyzedinfant formula. EPI pigs were 10 weeks of age (+/−2 weeks), whichcorresponds to about 6 months of age for a human infant. Pigs were feedNestle (NAN Pro 1 Gold) infant formula enriched with 2% ARAtriglycerides (TG-ARA) and 1% DHA triglycerides (TG-DHA) from fish oil(NuCheck (http://www.nu-chekprep.com, ˜40% TG-DHA and TG-ARA). Feedingoccurred every 3 hours, 4 times per day. In the group of pigs receivingpre-hydrolyzed formula, the formula was pre-hydrolyzed 15 minutes beforefeeding by mixing with CV lipase or RO lipase at 37° C. The duration ofthe experiment was 1 week, followed by analysis of LC-PUFA concentrationin faeces, absorption of LC-PUFA in plasma, and accretion of LC-PUFA intissues (retina, heart, liver, kidney, erythrocytes, brain, and fat).

As shown in FIG. 22A, EPI pigs given formula pre-hydrolyzed with CVlipase or RO lipase had significantly reduced stool weight (CV: >60%reduction, p<0.001; RO: ˜30% reduction, p<0.05). Pre-hydrolysis of fatwith CV lipase or RO lipase also significantly reduced total fat contentin the faeces compared to control EPI pigs (FIG. 22B) and significantlyincreased the coefficient of fat absorption (% CFA) compared to controls(FIG. 22C), where % CFA=(fat intake (g/24 hr)−fat in faeces (g/24hr))/(fat intake (g/24 hr)), n=3/arm, p=0.002 for CV vs. control, andp=0.003 for RO vs. control. As compared to control, pre-hydrolysis witheither CV lipase or RO lipase caused significant reductions in faecalARA, (36% and 65% reductions, respectively), EPA (78% reduction witheither enzyme), and DHA (68% and 60% reductions, respectively) (FIGS.23A-23C). These data show that pre-hydrolysis of formula with CV or ROlipase reduces faecal levels of total fat, ARA, DHA, and EPA, indicatingan improvement in the absorption of omega-3, omega-6, and total fattyacids.

In addition, pigs fed pre-hydrolyzed formula had a significant increasesin plasma and tissue levels of ARA and DHA after 7 days of feedingcompared to control pigs. For these studies, there were 4 pigs in theControl and CV lipase groups and 3 pigs in the RO lipase group.Pre-prandial plasma samples were taken after overnight fasting following7 days of treatment. Plasma levels of ARA and DHA were significantlyhigher (60% and 30%, respectively, p<0.05) in pigs fed formulapre-hydrolyzed with RO lipase compared to pigs fed non-hydrolyzedformula (FIGS. 24A and 24B). Plasma levels of ARA were alsosignificantly higher (40%, p<0.05) in pigs fed formula pre-hydrolyzedwith CV lipase compared to pigs fed non-hydrolyzed formula (FIGS. 24Aand 24B). ARA and DHA levels also increased significantly (p<0.05) inthe retina (FIG. 25A) and adipose tissue (FIG. 25B) of pigs fed formulapre-hydrolyzed with RO lipase compared to pigs fed non-hydrolyzedformula. Retina levels of ARA were also significantly higher (p<0.05) inpigs fed formula pre-hydrolyzed with CV lipase compared to pigs fednon-hydrolyzed formula (FIG. 25A). In pigs fed formula pre-hydrolyzedwith CV or RO lipase, ARA levels also increased significantly (p<0.05)in the heart (FIG. 26A: 60% and 20% increase, respectively) and kidney(FIG. 26B) of pigs compared to pigs fed non-hydrolyzed formula. DHAlevels increased significantly (60%, p<0.05) in the heart of pigs fedformula pre-hydrolyzed with CV lipase compared to pigs fednon-hydrolyzed formula (FIG. 26A). There were minimal or no changes inliver, erythrocytes, and brain, which may be explained by the relativelyshort duration of treatment (7 days) in this study.

Example 8: Hydrolysis of TG-DHA and TG-ARA in Infant Formula byImmobilized Lipase in a “Teabag”

Rhizopus oryzae (RO) lipase was covalently bound to acrylic beads andcontained in a device resembling a teabag. Enfalac infant formula (25 g)was combined with tap water (88 mL) at 37° C. Reactions were carried outin a glass bottle with 100 mL of infant formula and a tea bag containingeither 100, 500, 1000, or 2000 mg of immobilized RO lipase. Eachreaction was incubated at 37° C. for 30 minutes with inversion. Sampleswere taken at the following timepoints: 0, 1, 2, 3, 4, 5, 10, 20, and 30minutes. Samples were analyzed for DHA and ARA by reverse phase highperformance liquid chromatography (RP-HPLC).

At each concentration of immobilized RO lipase, the percent hydrolysisof DHA and ARA increased as the amount of immobilized RO lipaseincreased (FIGS. 27A-27D). These data demonstrate the feasibility of thetea bag device for pre-hydrolyzing formula with lipase.

Example 9: Hydrolysis of TG-DHA and TG-ARA in Infant Formula byImmobilized Lipase in a Cartridge

Rhizopus oryzae (RO) lipase and Chromobacterium viscosum (CV) lipasewere immobilized onto macroporous acrylic polymer beads (Immobeads™;ChiralVision). Approximately 200 mg of RO lipase were used per gram ofbeads. A sample of CV lipase-coated beads was irradiated (CVI) todetermine the effect of irradiation on potency of immobilized lipase.Approximately 1.7 g of each bead preparation (RO, CV, and CVI) werepacked into columns with bed volumes of approximately 5 mL. Infantformula containing DHA and ARA triglycerides was passed over the columnat a flow rate of 75 mL/hr. The column eluate was analyzed for DHA andARA hydrolysis by HPLC. The percent hydrolysis of DHA and ARAtriglycerides by CV, CVI, and RO lipases is shown in Table 6.

TABLE 6 Hydrolysis of TG-DHA and TG-ARA using immobilized lipasecartridge Column % Hydrolysis % Hydrolysis packing TG-DHA TG-ARA CV 5 mL92.50 41.00 CVI 5 mL 71.90 34.50 RO 5 mL 98.79 94.85

Example 10: RO Lipase Versus Pancreatin

Rhizopus oryzae (RO) lipase displayed far greater activity toward DHAand ARA triglycerides than porcine pancreatin (Zenpep®), which containsa mixture of pancreatic lipases, proteases, and amylases. 1.4 mL ofinfant formula was mixed with 100 uL of lipase (either pancreatin or ROlipase) and 100 uL each of triglycerides of DHA and ARA. Reactions wereincubated at 37° C. for 15 minutes. Samples were taken at time points 0,1, 2, 4, 6, 8, 10, and 15 minutes and analyzed by RP-HPLC for DHA andARA. DHA (FIG. 28A) and ARA (FIG. 28B) triglycerides were hydrolyzed byRO lipase over time but were not hydrolyzed by pancreatin.

Example 11: 6-Week Pig Study: Long-Term Feeding of Pigs with FormulaPre-Hydrolyzed by Immobilized Lipase

Six healthy pigs and twenty pigs with surgically-induced exocrinepancreatic insufficiency (EPI) (see Example 5) were subjected to atwo-week adaptation/control period followed by a six-week testing period(FIG. 29). During the adaptation period, all pigs were fed NAN Pro 1Gold infant formula (Nestle) (“IF”). During the testing period, thehealthy pigs (“Healthy”) and six of the pigs with EPI (“EPI”) were fedinfant formula fortified with TG-LCPUFA (see Examples 5 and 6); seven ofthe pigs with EPI were fed TG-LCPUFA-fortified infant formula that hadbeen pre-hydrolyzed with immobilized RO enzyme using a “teabag” device(“EPI+iRO”). The remaining pigs were withdrawn from the study forvarious reasons, e.g., failure to surgically induce complete EPI.

For pre-hydrolysis, 2 liters of NAN Pro 1 Gold (Nestle) infant formulawere prepared by mixing 1.5 liters of water at 37 C with 500 g ofpowdered formula fortified with 50 mg/kg TG-DHA and 100 mg/kg TG-ARA(see Example 5, Section 5.3). Five teabag-like devices containing ROlipase immobilized on beads (1 gram of immobilized lipase in each“teabag”) were added to the 2 liters of formula and mixed at roomtemperature for 15 minutes using a magnetic stirrer at constant mixingspeed. This corresponds to 9,000 units (as measured against olive oil)of immobilized RO lipase per 150 grams of total fat in the fortifiedformula (60 U/g total fat). Before hydrolysis, the fortified formulacontained 17.4 mmol/liter of non-esterified fatty acids. Afterhydrolysis, the formula contained 107.6 mmol/liter of non-esterifiedfatty acids.

Food consumption was measured daily. Blood and stool samples werecollected at the end of the adaptation period (“basal”), and after weeks1, 4, and 6 of the treatment period. For the basal sample, faeces werecollected for 48 hours (2×24 h). For the week 1, 4, and 6 samples,faeces were collected for 72 hours (3×24 h). At the completion of thetreatment period, organs and tissues were collected for absorption andsafety studies.

Pre-hydrolyzed formula was well tolerated with no treatment-relatedchanges in food intake, growth, organs (by gross examination), orgeneral well being. There was no fatty liver development based on grossliver examination when the pigs were sacrificed at the end of the 6-weekstudy.

After six weeks, there was a statistically-significant increase in ARA(FIG. 30A) and DHA levels (FIG. 30B) in erythrocytes (20% and 36%,respectively) in EPI pigs fed pre-hydrolyzed formula (EPI+iRO), ascompared to EPI pigs fed the TG-LCPUFA-fortified formula withoutprehydrolysis (EPI). Erythrocyte levels of ARA and DHA did not differsignificantly between healthy pigs and EPI pigs fed pre-hydrolyzedformula for six weeks.

As shown in Table 7, there was a statistically-significant increase inplasma levels of triglycerides, cholesterol, HDL, and LDL in EPI pigsfed pre-hydrolyzed formula for six weeks. Plasma levels oftriglycerides, cholesterol, HDL, and LDL did not differ significantlybetween healthy pigs and EPI pigs fed pre-hydrolyzed formula for sixweeks.

TABLE 7 Group Triglycerides Cholesterol HDL LDL Healthy 4.13 ± 0.68 0.51± 0.25 2.04 ± 0.31 1.27 ± 0.33 EPI 2.69 ± 0.56 0.22 ± 0.07 1.46 ± 0.410.69 ± 0.35 EPI + iRO  4.09 ± 1.47*  0.44 ± 0.19*  1.93 ± 0.46*  1.13 ±0.54*(All values in mmol/L. Asterisks indicate p<0.05 for the differencebetween EPI and EPI+iRO.)

As shown in Table 8, pigs fed pre-hydrolyzed formula for six weeks hadincreased plasma levels of vitamins A and E, but no significantdifference for vitamin D. There was a statistically significantdifference (p<0.05) in the plasma level of vitamin E between the EPI andEPI+iRO groups. For vitamin A, there was a statistically significantdifference (p<0.05) between the EPI and healthy groups but not betweenthe EPI+iRO and healthy groups.

TABLE 8 Alpha-tocopherol Retinol 25-OH-D3 mg/mL mg/mL ng/mL GroupVitamin E Vitamin A Vitamin D Healthy 6.6 ± 1.4 0.34 ± 0.06  9.4 ± 2.0EPI 0.8 ± 0.4 0.18 ± 0.06 10.4 ± 3.6 EPI + iRO  1.5 ± 0.9* 0.26 ± 0.1710.2 ± 4.1

1-89. (canceled)
 90. A method of providing a nutritional formula to apatient, at a point of care, the method comprising: receiving anutritional composition within a point-of-care device, the point-of-caredevice comprising: a container for receiving the nutritionalcomposition; and a lipase immobilized by covalent binding to a structurethat is in fluid communication with the container; exposing, at thepoint of care, the nutritional composition to the immobilized lipasewithin the point-of-care device to hydrolyze long-chain polyunsaturatedfatty acid (LC-PUFA) triglycerides and/or LC-PUFA esters within thenutritional composition into monoglycerides and free fatty acids to formthe nutritional formula; and providing the nutritional formulacomprising monoglycerides and free fatty acids to the patient foringestion.
 91. The method of claim 90, wherein the nutritionalcomposition is exposed to the lipase for at least one minute, prior toingestion by the patient.
 92. The method of claim 90, wherein thenutritional composition is exposed to the lipase for no more than thirtyseconds, prior to ingestion by the patient.
 93. The method of claim 90,wherein the nutritional composition is exposed to the lipase for no morethan one minute, prior to ingestion by the patient.
 94. The method ofclaim 90, wherein the nutritional composition is exposed to the lipasefor no more than five minutes, prior to ingestion by the patient. 95.The method of claim 90, wherein the nutritional composition is exposedto the lipase for no more than sixty minutes, prior to ingestion by thepatient.
 96. The method of claim 90, wherein the nutritional formula isprovided to the patient before re-esterification of the free fattyacids.
 97. The method of claim 90, wherein the structure comprisesparticles contained within the container, and wherein exposing thenutritional composition to the immobilized lipase comprises moving thenutritional composition through the particles contained within thecontainer.
 98. The method of claim 90, further comprising retaining theimmobilized lipase within the point-of-care device while allowing thenutritional formula to exit the point-of-care device.
 99. A method ofproviding a nutritional formula to a patient, at a point of care, themethod comprising: passing a nutritional composition comprisingtriglycerides into a handheld device; exposing the nutritionalcomposition to lipase immobilized by covalent binding to a solidstructure within the handheld device, wherein exposing the nutritionalcomposition to the lipase hydrolyzes at least some of the triglyceridesto form monoglycerides and free fatty acids to create the nutritionalformula; and delivering the nutritional formula from the handhelddevice.
 100. The method of claim 99, further comprising providing thenutritional formula to the patient.
 101. The method of claim 100,wherein the nutritional formula is provided to the patient beforere-esterification of the free fatty acids.
 102. The method of claim 99,wherein the nutritional composition is exposed to the lipase for atleast one minute, prior to ingestion by the patient.
 103. The method ofclaim 99, wherein the nutritional composition is exposed to the lipasefor no more than thirty seconds, prior to ingestion by the patient. 104.The method of claim 99, wherein the nutritional composition is exposedto the lipase for no more than one minute, prior to ingestion by thepatient.
 105. The method of claim 99, wherein the nutritionalcomposition is exposed to the lipase for no more than five minutes,prior to ingestion by the patient.
 106. The method of claim 99, whereinthe nutritional composition is exposed to the lipase for no more than 60minutes, prior to ingestion by the patient.
 107. The method of claim 99,wherein exposing the nutritional composition comprises passing thenutritional composition through a container of the handheld device thatcontains the solid structure to which the lipase is covalently bound.108. The method of claim 99, wherein the structure comprises a pluralityof particles, and the nutritional composition is passed through theplurality of particles.
 109. The method of claim 99, further comprisingretaining the immobilized lipase within the handheld device.
 110. Amethod of providing a nutritional formula to a patient, at a point ofcare, the method comprising: receiving a nutritional compositioncomprising long-chain polyunsaturated fatty acid (LC-PUFA) triglyceridesinto a point-of-care device; exposing the nutritional composition tolipase immobilized by covalent binding to a structure of thepoint-of-care device, wherein exposing the nutritional composition tothe lipase hydrolyzes at least some of the LC-PUFA triglycerides to forma nutritional formula comprising monoglycerides and free fatty acids;and providing, at the point of care, the nutritional formula to thepatient.
 111. The method of claim 110, further comprising retaining theimmobilized lipase within the point-of-care device so that no more than0.1 milligram of lipase per milligram of LC-PUFA is delivered from thepoint-of-care device.
 112. The method of claim 110, wherein exposing thenutritional composition to lipase causes the nutritional formula tocomprise more free fatty acids than monoglycerides.